Immunocytochemical localization of transient DNA strand breaks in differentiating myotubes using in situ nick-translation

DEVELOPMENTAL

BIOLOGY

127,362-367 (1988)

lmmunocytochemical Localization of Transient DNA Strand Breaks in Differentiating Myotubes Using in Situ Nick-Translation BARBARA A. DAWSONANDJOHNLOUGH Department

of Anatomy

and Cellular Biology, Medical College of WisconGn, Milwaukee, Accepted February

Wisconsin 53226

16, 1988

We have localized DNA strand breaks during in vitro chicken myogenesis by repairing nicks in nuclei of fixed cell monolayers in situ with biotin-11-dUTP, followed by immunocytochemical detection of incorporated biotin with rabbit anti-biotin and FITC-labeled goat anti-rabbit antibodies. No accumulations of biotin sufficient for immunocytochemical detection were observed in 23-hr cultures of dividing cells. In 33- and 43-hr cultures, biotin was first detected in only 3% of the nuclei, all of which appeared to be in fusing myoblasts or small myotubes. In contrast, cultures of young, highly fused myotubes (56 hr) exhibited 18% biotinylated nuclei; virtually all of these nuclei, most of which were grouped as aggregates, were within myotubes. In older cultures (73 and 94 hr) incorporation of biotin into myotube nuclei markedly decreased, while increases were noted in nuclei of mononuclear cells. These results indicate that extensive single-stranded DNA nicking occurs in nuclei of young myotubes, followed by repair as terminal differentiation

eIlSUeS.

0 1988 Academic

Press, Inc.

INTRODUCTION

Chromosome breakage has been identified as a normal, developmentally regulated process in lower eukaryotic organisms including the ascarid worm (review, Wilson, 1928; Moritz and Roth, 1976) and ciliated protozoa (Prescott and Murti, 1973; Yao, et ab, 1987). Breakage of chromosomes may also be an obligatory feature of vertebrate cell differentiation. Using physical techniques to assess the integrity of total DNA in cultures of murine erythroleukemia cells, Terada et al, (1978), Scher and Friend (1978), and McMahon et al. (1984) have demonstrated the occurrence of single-stranded breaks during induction of cytodifferentiation. Singlestranded DNA breaks have also been observed during the differentiation of human promyelocytic leukemia cells (Farzaneh et al., 1987), human peripheral blood lymphocytes (Johnstone and Williams, 1982) and, most pertinent to this study, chicken embryo skeletal muscle cells (Farzaneh et aZ.,1982). During the differentiation of cultured chicken skeletal muscle cells, myoblasts divide for approximately 35 hr, irreversibly withdraw from the cell cycle, and fuse to form multinucleate myotubes. Myotube formation, which heralds the onset of terminal differentiation, is essentially complete early in the third day of culture at which time approximately 70% of the nuclei in the culture are within myotubes. In their study, Farzaneh et al. (1982) utilized alkaline and neutral DNA sedimentation analyses to detect single-stranded nicks which appeared at the onset of myotube formation (44 hr). Since their determinations analyzed bulk DNA from a mixed 0012-1606/88 $3.00 Copyright All rights

0 1988 by Academic Press, Inc. of reproduction in any form reserved

population of cells-myoblasts, myotubes, and fibroblasts-within the myogenic cultures, it was not possible to ascribe breakage sites to individual cells. In the present study we have localized the cellular sites of DNA breakage during myogenesis using an in situ nick-translation technique in which a biotinylated nucleotide is incorporated into nicked strands and detected by immunofluorescent antibody staining. We report that accumulations of biotinylated DNA are first detected in a small percentage of fusing myoblasts (33-43 hr), followed by a peak of highly fluorescent sites in clustered myotube nuclei at the time of maximal fusion (56 hr). Detection of strand breaks in myotubes markedly declines as differentiation proceeds (73 and 94 hr). These findings have previously been reported in abstract form (Dawson and Lough, 1987). MATERIALS

AND METHODS

Materials Biotin-11-dUTP was purchased from BRL (Gaithersburg, MD). Escherichia coli DNA polymerase I was purchased from Molecular Biology Resources (Milwaukee, WI.). Rabbit anti-biotin and FITC-labeled goat anti-rabbit IgG were purchased from Enzo (New York, NY). [3H]dCTP was from ICN (Irvine, CA.), all other biochemicals were from Sigma (St. Louis, MO), and cell culture reagents were from GIBCO (Grand Island, NY). Cell Culture Myogenic cells were prepared from breast muscle of 12-day-old chicken embryos and plated at 75 X lo3 362

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cells/cm’ in gelatin-coated 35-mm culture dishes as previously described (Wunsch et al., 1987), except that cytosine arabinoside was used only where indicated (Fig. 2). Myoblast fusion was assessed as previously described (Lough and Bischoff, 1976). In Situ Nick-Transla,tion

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Determination

of DNA Content

The content of DNA in cultures was estimated by the Hoechst fluorometric method (Brunk et ah, 1979), using a Hoefer DNA TKO 100 Mini-Fluorometer. RESULTS

and Immunocytochemistry

At intervals during myogenesis, plates were removed and rinsed three times with minimum essential medium (MEM), followed by fixation with methanol:acetic acid (2O:l). After exactly 15 min at room temperature, the fixative was removed by rinsing three times with MEM and twice with 50 mM Tris . HCl (pH 7.5), 5 mM MgCIZ. To conserve reagents, nick-translations were performed in 25-~1 reaction mixture, which was uniformly spread across the cell monolayer by overlaying a 22 X 22-mm coverslip. The reaction mixture consisted of 50 mM Tris . HCl (pH 7.5); 5 mM MgClz; 10 mM 2-mercaptoethanol; 10 pg/ml acetylated bovine serum albumin (BSA, BRL); 5 PLM each dATP, dGTP, dCTP; and either TTP or biotin-11-dUTP, plus 200 U/ml DNA polymerase I. For kinetic determinations, [3H]dCTP (66 &i/ml; 13.2 Ci/mmole) was included in the reaction mixture. Reactions were terminated at the indicated intervals by floating off coverslips with ice-cold 10% trichloroacetic acid (TCA), followed by three 5-min changes with 10% TCA and two 5-min changes with ice-cold 100% EtOH; air-dried monolayers were solubilized in 1.0 N NaOH, neutralized with HCl, and counted in 10 ml BudgetSolve (RPI, Mount Prospect, IL). For immunocytochemical localization of sites incorporated with biotin-11-dUTP, the nick-translation was terminated by floating off coverslips with ice-cold phosphate-buffered saline (PBS), followed by seven changes of PBS at 5-min intervals. Thoroughly rinsed monolayers were blocked with PBS, 0.1% Triton X-100 for 2 min at room temperature, followed by three rinses with PBS. Four hundred microliters of rabbit anti-biotin, diluted 1:lOO in PBS containing 2 mg/ml BSA, was added to the dishes and incubated at 37°C in a humid atmosphere, After 60 min, the dishes were washed five times with ice-cold PBS, followed by a similar incubation with 400 ~1 FITC-labeled goat anti-rabbit antibody (1:lOO). Following thorough washing with ice-cold PBS, the monolayers were coverslipped with one or two drops of 10% PBS containing 90% glycerol and 0.2 Mn-propyl gallate (to reduce photobleaching; Giloh and Sedat, 1982) and examined with a Nikon Optiphot epifluorescent microscope (type B2 filter) equipped with a Microflex UFX-II photomicrographic attachment. The percentage of nuclei in each culture which exhibited immunofluorescence was determined by scoring at least 500 nuclei in random fields at a magnification of 400x.

The experiment shown in Fig. 1 was performed to assess the extent of endogenous DNA nicking in cultures between 18 and 90 hr of myogenesis. At all myogenie stages, the rate of [3H]dCTP incorporation during a 90-min nick-translation reaction was approximately linear (data not shown). Incorporation of label increased nearly sevenfold during the culture period (Fig. 1A). However, when normalized to DNA content in the cultures (Fig. lB), no marked changes in nicking of bulk DNA was detected, with the possible exception of a decrease at 66 hr of unexplained significance. All of the reactions in Fig. 1 were performed at a DNA polymerase I concentration of 200 U/ml reaction mixture. To ensure that these reactions were dependent upon DNA polymerase I and that this enzyme level was sufficient to nick-translate all DNA strand breaks in A

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FIG. 1. Extent of in situ nick-translation during myogenesis. Cultures at the indicated myogenic stages were nick-translated for 1 hr as described under Materials and Methods, using [“H]dCTP to monitor the reaction. The rate of reaction was essentially linear at all stages of myogenesis (not shown). Biotin-11-dUTP was not used in these reactions; DNA polymerase I was included at 200 units/ml reaction mixture. Reactions were terminated with 10% TCA and acid-insoluble radioactivity was determined. (A) cpm/35 mm culture dish; (B) cpm/pg DNA. The vertical bars indicate one standard deviation from the mean of triplicate determinations.

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the culture, the experiment shown in Fig. 2 was performed. In the absence of DNA polymerase I (0 U/ml), no incorporation of [3H]dCTP was detected. However, enzyme levels of 50-200 U/ml resulted in similar levels of incorporation, verifying the efficacy of the 200 U/ml concentration (open bars). In parallel cultures which had been treated with 10 PM cytosine arabinoside (araC; shaded bars) for 19 hr prior to nick-translation, the extent of DNA repair was observed to depend on DNA polymerase I levels between 50 and 200 U/ml. The fact that the latter concentration was sufficient to nicktranslate the increased numbers of strand breaks which accumulate during ara-C treatment further indicates the adequacy of the 200 U/ml enzyme concentration for cultures not treated with ara-C. The effect of ara-C, which is widely used to kill replicating cells in myotube cultures, has been consistently observed and is the focus of a separate study. Although the results of Figs. 1 and 2 established the time- and DNA polymerase I-dependence of the nicktranslation reaction, differences in the extent of DNAnormalized nick-translation were not apparent at any stage of myogenesis (Fig. 1B). This was unexpected, since the alkaline gradient results of Farzaneh et al. (1982) had revealed a precipitous onset of strand breakage at the time of myotube formation (44 hr). One explanation for this apparent discrepancy was that strand breakage might be facilitated by localized accumulations of nicks in selected nuclei. Thus, to determine the distribution and intracellular sites of DNA breaks, nick-translations were performed for 90 min with biotin-11-dUTP, followed by immunocytochemical localization of biotin. Although the substitution of biotinll-dUTP for TTP caused an approximate decrease of 25% in the overall reaction rate, linearity and time de-

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DNA Polymera~e I (mtsiml)

FIG. 2. DNA polymerase I dependence of in situ nick-translation. Cultures of myotubes (67 hr in vitro) were fixed and nick-translated with DNA polymerase I at the indicated levels. The extent of reaction after 90 min was determined as in Fig. 1; biotin-11-dUTP was not used. The shaded bars indicate values from cultures which were treated with 10 &’ ara-C for the 19-hr interval (48-67 hr in vitro) preceding harvest. Vertical bars indicate the range of duplicate determinations.

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Nuclei I” myotubes Labeled nuclei (total) Labeled nucla in myotubes

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FIG. 3. Percentage of nuclei immunostained for biotin in cultures at various stages of myogenesis. Open bars, percentage of nuclei in the culture which were in myotubes; shaded bars, percentage of nuclei in the culture which were immunofluorescently labeled. The hatched bars denote the percentage of biotin-labeled nuclei in the cultures which were within myotubes. Cultures were fixed, nick-translated with biotin-ll-dUTP for 90 min, and immunostained as described under Materials and Methods. Repetition of this experiment gave similar results.

pendence of the reaction were unaffected (data not shown). Figures 3 and 4 present the results of an experiment in which sites of extensive biotin incorporation, as revealed by immunostaining of nuclei, were enumerated at various myogenic stages. At each stage in Fig. 3, the extent of differentiation is indicated by myotube formation (open bars). The shaded bars indicate the percentage of nuclei in the culture which exhibited immunostaining for biotin; a nucleus was considered stained only if it contained punctate immunofluorescence which was clearly within the confines of the nuclear membrane. Cultures of dividing myoblasts (23 hr), in which as many as 30% of the cells may be in S phase at one time (Lough and Bischoff, 1976), exhibited no biotin staining. This was unexpected, since the experiment shown in Fig. 1 indicated that these cells contain nicked DNA. Our interpretation of this finding is that dividing cells incorporate nucleotides into randomly dispersed replication forks, resulting in a diffuse incorporation of biotin which is insufficient for detection at the level of immunocytochemical light microscopy; validation of this interpretation will require quantitative immunogold electron microscopy (see below). In 33- and 43-hr cultures, when cells are withdrawing from the cell cycle and beginning to form myotubes, biotin was detected in only 3% of nuclei, all of which appeared to be in fusing myoblasts (33 hr) or small myotubes (43 hr; hatched bars). In contrast, when fusion was maximal at 56 hr, biotin was detected in 18% of the nuclei in the culture; virtually all stained nuclei, most of which were arranged as aggregates, were within myotubes. At later

DAMON

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stages of myogenesis (73 and 94 hr), biotin labeling of myotube nuclei sharply declined. Upon repeating this experiment we determined a duplicate mean value of 24% stained nuclei at maximum fusion, followed within 7 hr by a decline to only 2% labeling. Most of the biotin detected in older cultures was in nuclei of mononuclear cells. Although this phenomonen is currently unexplained, it could represent an early step in the commitment of these cells to eventually fuse. Figure 4 presents photomicrographs of these immunostained cells at 56, 73, and 94 hr of differentiation. The 56-hr myotube indicated by the arrows in the phase-contrast image (Fig. 4A) contained an aggregate of approximately 25 biotin-labeled nuclei (Fig. 4B); the absence of nuclear staining in other myotubes in this microscopic field suggests that nicking is restricted to subpopulations of myotube nuclei. Figure 4C is a higher magnification of a separate 56-hr myotube which also

365 contained a large aggregate of labeled nuclei. In contrast, the 73-hr myotubes shown in Figs. 4D-4F displayed few, if any, labeled nuclei (the arrowheads point to intensely fluorescent extracellular debris). And, Figs. 4G and 4H show a field containing one of the few 94-hr myotube nuclei which exhibited immunostaining; most of the biotin label in this culture was in mononuclear cells. As a control to verify that labeling was dependent upon biotin incorporation, the cells in Fig. 41 were nick-translated without biotin-11-dUTP. Morphological features which are difficult to discern in Fig. 4 are of additional interest. First, phase-contrast images of biotinylated nuclei were morphologically indistinguishable from nuclei with no biotin. Thus, although artifacts cannot be completely ruled out in mediating this phenomenon, there is no evidence that nucleotide was selectively incorporated into moribund myotube segments. Furthermore, the likelihood that in

FIG. 4. Microscopic localization of nick-translated sites after immunostaining. (A, D, G) Phase-contrast images. (B, C, E, F, H, I) Immunofluorescent images. Myotube cultures were fixed, nick-translated, and immunostained as described under Materials and Methods. A 56-hr myotube containing a cluster of approximately 25 biotin-labeled nuclei is depicted by the arrows in (A) and (B). (C) A higher magnification of a separate 56-hr myotube containing a cluster of biotinylated myonuclei. At 73 hr, myonuclei exhibited little, if any, biotin staining: ((D) and (E) depict the same fields; (F) is a different field at the same magnification). (G, H) Matched fields from a 94-hr culture; (H) Diffuse biotin deposits, mostly in mononuclear cells. (I) A control which was nick-translated without biotin-ll-dUTP; staining was not detected in any nuclei in the culture. Arrowheads point to intensely fluorescent debris in the cultures. Scale bars = 9.3 pm.

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situ nick-translation detects DNA segments undergoing breakage and religation is further supported by our observations that treatment of myotubes with ara-C, an inhibitor of DNA repair, results in significantly higher numbers of biotinylated nuclei (not shown). Regarding the staining pattern, it is noted that the pun&ate fluorescence was not restricted to any specific region of the nucleus. However, these nick-translations were performed on whole cells; discrete intranuclear localization of strand breaks must be performed on sectioned nuclei to avoid artifacts associated with the limited diffusion of reactants. Thus, we plan to assess intranuclear sites of strand breakage using indirect immunogold electron microscopy. On the other hand, visualization at the light microscopic level did reveal that staining was always excluded from the nucleolus. Finally, incubation with 0.5-1000 rig/ml DNase I prior to biotin incorporation caused all nuclei in the culture to exhibit diffuse staining which increased in intensity as a function of DNase I concentration. DISCUSSION

During the past decade, density gradient sedimentation analysis has revealed that the differentiation of eukaryotic cells is accompanied by breakage of DNA (Terada et al., 1978; Scher and Friend, 1978; Farzaneh et al., 1982; Johnstone and Williams, 1982; Farzaneh et al., 1987). The study of Farzaneh et al. (1982) demonstrated extensive single-stranded DNA breaks at the onset of myotube formation during myogenesis. In this study, we have extended these findings, providing immunocytochemical evidence that the major intracellular site of DNA strand breakage during myogenesis is in clusters of nuclei within nascent myotubes. The fact that this phenomonen (a) in 33- and 43-hr cultures is detected only in fusing myoblasts and myotubes, (b) is most pronounced at the time of maximum fusion (56 hr), and (c) is transient, suggests that nicking is restricted to fusing or recently fused nuclei, followed by repair as myotubes commence terminal differentiation. Our data are consistent with evidence implicating a requirement for DNA repair at the onset of cytodifferentiation. A well-documented response to DNA strand breaks is the activation of the enzyme poly(ADP-ribose) synthetase (pADPRS), which binds DNA near breakage sites and transfers poly(ADP-ribose) moieties from NAD to proteins (reviews, Ueda and Hayaishi, 1985; Gaal and Pearson, 1986). Although the nuclear function of pADPRS has not been fully clarified, it is involved in DNA repair, possibly by regulating the activity of DNA ligase (Ohasi et al., 1983). The singlestranded DNA breaks observed during human promyelocytic leukemia cell induction (Farzaneh et cd, 1987),

VOLUME 12’7,1988

human peripheral blood lymphocyte activation (Johnstone and Williams, 1982), and skeletal myogenesis (Farzaneh et al., 1982) are accompanied by increases in pADPRS activity which are obligatory for these cells’ differentiation. In each case, treatment with S-aminobenzamide, an inhibitor of pADPRS activity, prevents cytodifferentiation. Moreover, Cherney et al. (1985) recently reported a threefold, transient increase in pADPRS activity as limb mesenchymal cells initiate terminal differentiation into skeletal muscle and cartilage cells. The control and specificity of DNA breakage during differentiation remains unknown. In this regard, endogenous nucleases have been identified which selectively degrade transcriptionally active genes (Vanderbilt et al., 1982; Anderson et al., 1986; Villeponteau et al., 1986). Recently, a DNA-binding protein which induces differentiation in myeloid hemopoietic cells has been shown to cause single-stranded nicks in doublestranded DNA (Weisinger et aZ., 1986). And, Yao et al. (1987) have identified a conserved 15-nucleotide sequence which is exclusively associated with chromosomal breakage sites during Tetrahymena development. Taken together, these findings provide strong evidence that the process of eukaryotic cytodifferentiation requires an orderly sequence of DNA breakage and repair, the significance of which is unknown. It can be speculated that these events are indicative of chromatin remodeling which results in the reposturing of genes which must be stably expressed to maintain the differentiated state. In this regard, Hutchison and Weintraub (1985) have presented evidence indicating that actively transcribed genes are nonrandomly situated in the nucleus. Therefore, using an avidin affinity chromatography technique that we have recently developed to isolate biotinylated sequences enriched for transcriptionally active genes (Roseman et al., 1986; Dawson et al., 1987), we are investigating the intriguing possibility that the biotinylated DNA sequences in myotube nuclei are contiguous with muscle-specific genes which are expressed during terminal differentiation. This work was supported by NIH grant HD 20743. B.A.D. is a Predoctoral Fellow of the American Heart Association, Wisconsin Affiliate. We thank Drs. Earl Godfrey and Tim Herman for helpful comments regarding the manuscript, and Mary H. Parlow and Theresa Miresse for expert technical assistance. REFERENCES ANDERSON, 0. D., Yu, M-M., and WILT, F. (1986). Site and stage specific action of endogenous nuclease and micrococcal nuclease on the histone genes of sea urchin embryos. Dev. Bid. 117,109-113. BRUNK, C. F., JONES, K. C., and JAMES, T. J. (1979). Assay for nanogram quantities of DNA in cellular homogenates. Anal. B&hem. 92,497-500.

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DNA Strand

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