0 1987 Alan R.Liss, Inc.

Cytometry 8:138-145 (1987)

Application of Pyronin Y(G) in Cytochemistry of Nucleic Acids' Zbigniew Darzynkiewicz, Jan Kapuscinski, Frank Traganos, and Harry A. Crissman Sloan-Kettering Institute for Cancer Research, Walker Laboratory, Rye, New York 10580 (Z.D., J.K., F.T.) and Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (H.A.C.) Received for publication July 31, 1986;accepted October 16, 1986.

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y (PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation. The products of PY interaction with doublestranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 pM, the RNA stainability decreases, perhaps

due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in 6 2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared. Key terms: RNA content, RNA conformation, nuclear RNA, flow cytometry, CHO cells, mitosis

In an accompanying paper (171, we describe biophysical and biochemical studies on pyronin Y (PY) and interactions of this dye with natural and synthetic nucleic acids in solutions. Several observations from these studies could be of importance for the qualitative and quantitative application of PY, both as a n absorption dye for light microscopy (2,191 and as a fluorochrome (4,23,26,28). In the present paper, we demonstrate that the biophysical properties of the dye complexes with nucleic acids observed in solutions (17) are relevant in cytochemistry. The combined biophysical and cytochemical approach as described in this and the accompanying paper (17) makes it possible to better understand the complexity of the dye-nucleic acid interactions and define optimal conditions for use of this dye as a specific cytochemical probe of nucleic acids.

phase either as monolayer cultures, or in suspension culture, free of mycoplasma contamination, in Ham's F10 medium supplemented with 10% heat-inactivated newborn calf serum, as previously described (6).To obtain populations enriched in mitotic cells, the suspension cultures were treated with 0.1 pg/ml Colcemid (GIBCO, Grand Island, NY) for up to 3 hr; this resulted in a n increase in the proportion of mitotic cells from 2 to 20%. Alternatively, a n approximately 96%pure population of mitotic cells was obtained by selective cell detachment at mitosis (6,221. Cells were fixed in 70% ethanol at 0-4"C, and maintained in fixative for up to 14 days at 4°C. Nuclei from CHO cells were isolated according to the procedure described by Gurley et al. (131, employing the

MATERIALS AND METHODS

Supported by PHS grants CA 23296,CA 28704,and P41-RR01315. Address reprint requests to Dr. Zbigniew Darzynkiewicz, Sloan-Kettering Institute, Walker Laboratory, 145 Boston Post Road, Rye, NY 10580.

Chinese hamster cells (CHO, originally obtained from Dr. T.T. Puck) were maintained in exponential growth

APPLICATION OF PYRONLN IN NUCLEIC ACID CYTOCHEMISTRY

nonionic detergent Nonidet P-40 and sodium deoxycholate. The cells were washed with 0.25 M sucrose and suspended in 10 mM NaC1, 0.15 mM MgC12,lO mM Tris, pH 7.4 ( - lo7 cells per 4 ml, a t 0-4°C). This suspension was vortexed at maximum speed for 30 sec. After standing 5 min, 0.5 ml of 20% (vh) aqueous solution of Nonidet P-40(Sigma Chemical Co., St. Louis, MO) was added, and the suspension was vortexed as above. After a n additional 15 min, a n aliquot of 0.5 ml of 0.5% (w/v) aqueous solution of sodium deoxycholate (Sigma) was added, and the suspension was vortexed again. After standing another 15 min in the presence of Nonidet and deoxycholate, the suspension was vortexed once more. The nuclei were pelleted by centrifugation a t 1,000 g for 10 min and resuspended in Hanks’ balanced salt solution containing Ca2+ and Mg2+. All procedures were done a t 0-4°C.Observation of nuclei obtained by this technique in the phase contrast microscope failed to reveal any adhering cytoplasmic ‘ 30 min), especially a t room temperature, there is a n increase in cell heterogeneity with respect to their fluorescence and a change in their light-scatter properties as well. Another point worth mentioning is that, considering the cell number per sample and thus RNA and DNA content, the D/P ratio was well above 2, even at the lowest PY concentration. Thus, the observed increase in stainability is not simply a reflection of the availability of the ligand per binding site. Figure 6 illustrates changes in fluorescence as a result of RNA denaturation by heat. In this experiment, cells were fixed, treated with DNase I, heated a t various temperatures, then stained either with ethidium bromide (EB; 5.1 pM), or PY (6.6 pM), and the fluorescence measured by flow cytometry. EB is highly selective towards ds nucleic acids; it does not stain ss nucleic acids (201, and therefore was used as a control to indicate the

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stainability can be conveniently suppressed by another dye, such as methyl green (2,191. On the other hand, DNA denaturation results in its increased stainability with PY (19). Our present (17) and earlier (8) data provide a n explanation for the dye specificity toward RNA and describe the nature of the PY-nucleic acid complexes that can be visualized by light microscopy. Specifically, we observed that PY induces condensation of nucleic acids; the condensation is followed by agglomeration resulting in formation of precipitates, such as described by Scott (25). The appearance of the condensed particles representing PY-nucleic acid complexes can be monitored by light-scatter measurements (8).The highly cooperative curves of light-scatter increase allow accurate estimates of the specific critical concentration of the dye (Cc) a t which the condensation occurs. As shown in

APPLICATION OF PYKONIN IN NUCLEIC ACID CYTOCHEMISTRY

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izing the PY-nucleic acid complexes that can be detected by fluorescence are entirely different from the ones described for the products detected by light absorption. First of all, this staining is restricted to ds regions of RNA. As shown in the accompanying paper (17)for the free nucleic acids and in Figure 6 under in situ conditions, fluorescence of PY bound to ss regions of RNA is quenched. Thus, the staining reactions observed by fluorescence vs. light absorption are mutually exclusive; the first is related to ds RNA, whereas the latter represents PY complexes with ss RNA. With this knowledge, it is possible to explain results in the literature that appeared to be paradoxical to the authors who observed that PY stains DNA and not RNA in the cell (24). At the high PY concentration used in these studies (66 mM) cellular RNA is expected to be denatured since PY may denature RNA as does another intercalating dye, acridine orange (7,9). The complexes of PY with denatured RNA thus, while visible by light microscopy, cannot be detected by fluorescence. On the other hand, at so high a concentration, PY intercalates into DNA, and this product can be detected by fluorescence. Because only ds RNA regions fluoresce following staining with PY, and because ds RNA undergoes denaturation in the presence of a high concentration of PY, caution should be exercised in selecting optimal concentrations of this dye for cytochemical detection of RNA.

144

DARZYNKIEWICZ ET AL.

Table 2

Advantages and Limitations of the Techniques Using PY or A 0 for RNA Detection by Flow Cytometry

Hoechst 33342PY

Stoichiometryof RNA staining Sensitivity of RNA detection DNA measurement accuracy

Poor

Quantum yield, signal intensity DNA and RNA staining specificity Stringency requirements and complexity of the staining reaction Ability to detect changes in RNA conformation Excitation requirements

Low Good

A0 Good Low

High High

Fair; depends on cell type High

Average

Fair High

Yes

Yes

Two sources

Single illumination

of illumination

In light of the above specificity for ds RNA, the results showing a decrease in stainability of RNA in mitotic cells as compared to cells in Gz phase are very interesting (Fig. 5). This data can be interpreted as indicating that, during transition of cells from Gz to M, there is a change in conformation of significant portions of cellular RNA from the ds- to the ss- form. It is known from biochemical studies that polyribosomes undergo dissociation a t the time of cells’ entrance to mitosis and that during mitosis, single ribosomes prevail (12). It is very likely, therefore, that PY detects a change in RNA conformation associated with this transition. Perhaps the RNA of single ribosomes in contrast to polyribosomes has a conformation that is either recognized by PY as a n ss- form or is more easily denatured by PY, In any event, the proportion of ds RNA stainable with the dye is decreased in M as compared to Gz cells. Thus, PY can be used as a probe of RNA conformation, e.g., to detect changes in RNA that occur during the cell cycle. Considering that fluorescence of PY is quenched by guanine (171, all the above discussion about stainability of ds RNA refers to RNA sections poor in G-C sequences. This base-specific quenching of PY fluorescence obviously restricts application of this dye even for quantitative analysis of ds RNA by fluorometry. First of all, stoichiometry of RNA staining cannot be achieved since the base sequences of the dye binding site have different effects on the quantum yield of the bound dye (17). Furthermore, even comparison of relative fluorescence intensities of cells cannot provide accurate information on relative RNA content between the cells if these cells contain RNA of different base sequence and/or composition.

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33342’py Method for DNA and RNA Detection The combination of PY with Hoechst 33342 provided a method for simultaneous, correlated measurements of RNA and DNA in flow cytometry (26). The alternative method to analyze these entities by flow cytometry is based on the use of the metachromatic dye, acridine orange (11).Each method has limitations and advantages (Table 2). The main limitation of the PY-Hoechst 33342 technique is a lack of correlation between RNA

content and fluorescence intensity. In contrast, the AORNA complexes have 1:l (DP)stoichiometry (18),and correlation between AO-fluorescence and RNA is linear over a wide range of RNA content within cells (1).On the positive side, a combination of Hoechst 33342 with PY, employing two-laser excitation, offers nearly a total separation of the DNA vs. RNA-associated emission spectra (4). This allows measurement of small quantities of RNA under conditions of high DNA/RNA ratios (e.g., as in isolated nuclei) without a problem of spectrum overlap from DNA. Even with optimal selection of the emission filters, the spectrum overlap limits the minimal estimate of RNA by techniques using AO. Thus, provided that a strong excitation source (laser) is used, PY offers higher sensitivity of RNA detection compared to AO. However, because of rather low quantum yield of the PY-RNA complexes, the weak mercury lamp excitation source may be inadequate to obtain absolute fluorescence intensity high enough to measure low RNA content, even if the overlap from DNA- fluorescence is minimal. Another advantage of the combination of Hoechst 33342-PY is higher specificity of these dyes to nucleic acids compared to AO. Thus, in situations where cells contain glycosaminoglycans (heparin, hyaluronic acid) that stain with AO, the Hoechst 33342-PY technique may be advantageous. The higher specificity of Hoechst 33342 for DNA is also reflected by more accurate DNA measurements, as seen by comparing the coefficient of variation of the mean values of DNA content of GI cells stained with Hoechst 33342 and AO. The AO- technique (7,10,11,29) requires very stringent conditions of staining; even minor changes in A 0 concentration, or breakdown of equilibrium with the dye, can result in loss of specificity of staining of RNA vs. DNA. This is certainly a limitation. Although stainabilitv of nucleic acids with PY is also sensitive to the dye concentration (Fig. 41, the sensitivity is lower than in the case of AO. The maximal sensitivity for detecting changes in RNA conformation, e.g., as evidenced by the differential stainability of M vs. GZcells, is observed at 6.6 pM PY concentration. At PY concentration < 2 pM, the staining is stable and shows little sensitivity with respect to changes in RNA conformation. This low concentration of PY is, therefore, u

APPLICATION OF PYRONTN IN NUCLEIC ACID CYTOCHEMISTHY

recommended for quantitative analysis of ds RNA content by flow cytornetry. Both techniques can be used to assay RNA conformation; in the case of AO, it requires special ionic conditions ( M g 2 + ) to preclude RNA denaturation otherwise induced by A 0 (10). It should be stressed that the present studies and the discussion are limited to situations in which permealized, fixed cells are stained with PY. Viable cells also accumulate PY in mitochondria (31, and the extent of the dye binding to mitochondria vs. RNA varies, depending on PY concentration (8). Thus, use of PY as a probe of RNA in viable cells is inappropriate.

ACKNOWLEDGMENT We thank Ms. Rose Vecchiolla for typing the manuscript.

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