Differential effects of ethidium bromide on mitochondrial and nuclear DNA synthesis in vivo in cultured mammalian cells

Experimental Cell Research 72 (1972) 21 l-222

DIFFERENTIAL EFFECTS OF ETHIDIUM BROMIDE ON MITOCHONDRIAL AND NUCLEAR DNA SYNTHESIS IN VIVO IN CULTURED MAMMALIAN CELLS MARGIT Department

of Therapeutic

M. K. NASS

Research, University of Pennsylvania Philadelphia, Pa 19104, USA

School

of Medicine,

SUMMARY Ethidium bromide (EB), at concentrations of 0.1 to 5 ,ug/ml, was found to inhibit cell growth and mitochondrial DNA synthesis of cultured L cells (mouse) and BHK cells (hamster), as indicated by cell counts and the incorporation of 3H-thymidine per pg purified DNA. In contrast, nuclear DNA synthesis of L cells, BHK and polyoma virus transformed BHK cells was actually stimulated to up to 250 % of control values by treatment of cells with 0.1 to 2 pg/ml EB for 1 to 2 days. Double-labeling experiments were performed to follow the differential synthesis of mitochondrial and nuclear DNA synthesis and the decay of radioactivity in pre-existing DNA during up to 4 days of treatment with EB. These results and analytical determinations of mitochondrial DNA showed a slightly lower yield of mitochondrial DNA after 4 days of treatment with EB than in controls. The content of protein in mitochondrial fractions was increased to up to 190 % of control values. In all three cell lines EB treatment led to a structural alteration of covalently closed mitochondrial DNA, consisting in part of a change to an increased degree of supercoiling and in addition breakage of circular DNA without re-closing. This change involved the preexisting as well as the traces of newly synthesized DNA. These changes were reversible by subsequent growth of cells in EB-free medium.

The consequences of treatment of eukaryotic cells with the phenanthridine dye ethidium bromide (EB) are manifested primarily in the mitochondria. Exposure of facultative anaerobic yeast cells to this DNA intercalating dye leads to a quantitative conversion from wild type to the cytoplasmic petite mutation characterized phenotypically by a respiratory deficiency [l]. The mutation may be permanent or it is reversible at elevated temperatures [2]. In obligate aerobic yeast EB was reported to inhibit the synthesis of cytochromes a i-a,, b and c, but not to induce

the petite mutation [3]. In higher cells, our studies [4] with mouse L cells in culture have shown that in the presence of EB the concentrations of cytochromes a +a, and b decreased, a +a, more rapidly than b. The concentration of cytochromes c1 and c, on the other hand, increased or remained the same as in control cells. These changes were accompanied by an enlargement of mitochondria and a drastic reduction and abnormal arrangement of cristae. A second more normally organized population of mitochondria was apparent after prolonged exposure Exptl

Cell Res 72

212 Margit M. K. Nass to EB. The effects of EB were reversible at normal temperatures. Treatment of a green and a bleached mutant strain of Euglena gracilis also led to great abnormalities in mitochondrial (but not chloroplast) morphology, although manifested differently than in L cells [5]. We have shown previously two effects of EB on mitochondrial DNA [6, 71. First, the synthesis of mitochondrial DNA is selectively inhibited by EB whereas the synthesis of nuclear DNA appears stimulated. Second, the covalently closed circular DNA characteristic of mitochondria is structurally altered. Recent reports have shown that the synthesis of mitochondrial DNA of Physarum polycephalum [8], HeLa cells [9], and kinetoplast DNA of trypanosomids [lo] is selectively inhibited by EB. Furthermore, the in vivo effects of the drug include a change of superhelix density of mitochondrial DNA [l 11. EB was shown to inhibit preferentially the activity of partially purified mitochondrial DNA polymerase of rat liver as compared with the nuclear enzyme using the same DNA template [12]. In yeast some unique in vivo effects of EB on the structure of mitochondrial DNA have been reported. Dissecting the early stages of petite induction, parental DNA was found to become degraded [13, 141. Mutants containing DNA of reduced size [15] or seemingly lacking DNA [ 13, 161 have been isolated. Transient treatment of yeast with EB and subsequent growth without EB lead to the appearance of a mitochondrial DNA species with a lower than normal buoyant density [14]. Other effects of EB include the selective inhibition of mitochondrial RNA synthesis [17, 181 and protein synthesis [ 191 in HeLa cells. An increased affinity of the mitochondrial membranes for EB as a result of energy conservation has also been reported [20]. In this paper evidence is presented that Exptl

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mitochondrial DNA synthesis is selectively inhibited by EB in different mammalian cell lines. The fate of this DNA has been studied during prolonged (4 days) treatment with EB. Unlike some petite mutants of yeast, animal cells do not appear to lose mitochondrial DNA during EB treatment. The observations confirm and extend earlier ones [4, 6, 71. MATERIALS

AND METHODS

Cell cultures L cells (mouse fibroblasts) were grown in suspension culture in Joklik-modified medium GIBCo, Grand Island, N.Y.) with 10 % foetal calf serum. Antibiotics were added as described [6]. Freshly dissolved ethidium bromide, filter-sterilized, was added to some cultures. These and control cultures were kept in the dark. Polyoma virus-transformed baby hamster kidnev (BHK) cells were originally obtained by Dr R. Roosa. Wistar Institute. Philadeluhia. Thev were grown’ in monolayer culture in large roller -bottles

161.

All experiments were conducted with cells in the logarithmic phase of growth, either in suspension culture (L cells) or in non-confluent monolayers. All cell lines were examined for the absence of mycoplasma at monthly intervals and found to be negative.

Isolation of mitochondria The procedures were similar to those described [4, 211, with minor modifications. L cells were harvested by centrifugation and washed twice with PBS (0.16 M NaCl, 0.01-M phosphate buffer, pH 7.0). All manipulations were carried out at 4’C. The cell pellets (4 to 8 x lo8 cells) were suspended in 20 ml of cold 0.05 % bovine serum albumin in 25 mM Tris-HCl. pH 7.4, and 2 mM EDTA. The cells were disrupted in a Dounce. homogenizer, 20 ml of 0.6 M sucrose (in Tris-EDTA) was added, the cells were resuspended and mitochondria isolated by differential centrifugation (centrifugation twice at 500 g to remove nuclei and cell debris, then at 10000 g to sediment mitochondria). Residual nuclear DNA was removed by digestion with 50 pg/ml pancreatic DNase in 0.3 M sucrose, 5 mM MgCl for 30 min at room temperature. To stop the enzyme reaction mitochondria were chilled and EDTA was added to 0.02 M. The mitochondria were sedimented and washed in 0.3 M sucrose, 2 mM EDTA, 25 mM Tris buffer, pH 7.4. In some cases mitochondria were further uurified by centrifugation in a linear gradient of sucrose [21]. BHK and BHK-PyY cells were grown as monolayer cultures. The medium was decanted, the attached cells rinsed twice with TBS (0.15 M NaCl, 0.02 M Tris-HCl, pH 7.5) and removed from the .glass by

Effects of ethidium bromide on mitochondrial DNA synthesis addition of 10 ml of trypsin solution (0.25 % trypsin, 1 mM EDTA in TBS) for 2-3 min at 37°C in the roller apparatus. Fresh medium with serum was added and the cells collected, chilled, washed in PBS and processed further as described for L cells.

Isolation of mitochondrial nuclear DNA

213

900 CONTROL

DNA and

Mitochondrial pellets (fresh or frozen at - 110°C) were evenlv susoended in EST buffer (0.1 M EDTA 0.15 M N&l, 0.01 M Tris-HCl, pH 8‘.0), and lysed by adding an equal volume of 2 % SDS (sodium dodecyl sulfate) in EST for 10 min at room temperature. &aCl (5 I$ was added to a final concentr&ion of 1 M, the suspension was chilled for at least 1 h. then centrifuged at 17 000 g for 15 min. The super: natant solution was adjusted to contain 300 pg/ml EB (ethidum bromide) and CsCl (refractive index 1.3880 at 23°C) at a final volume of 5.0 ml. The samples were centrifuged to equilibrium in a Spinco SW65Ti rotor at 42 000 rpm for 44 to 48 h (or in some cases at 40 000 rpm for 63 h). The fluorescent bands of the gradients were examined in ultraviolet light and fractions were collected through the bottom of the tubes. The desired fractions of the gradients were freed of EB by dialysis or treatment with Dowex [22], concentrated if necessary by pervaporation in the cold, and redialyzed. Nuclear DNA was isolated by chloroform-octanol extraction of lysates (1 % SDS in EST) of purified nuclear fractions, followed by treatment with ribonuclease and equilibrium centrifugation in cesium chloride 1221.

Analytical techniques Chemical analyses of DNA and protein were performed as described previously [22]. For specific activity determinations DNA was analysed by absorption at 260 nm. Radioactive DNA was applied in 10 ~1 aliquots to Whatman no. 2 filter paper squares. The samples were immersed in 3 changes of ice-cold 5 % ?CA (tricarboxylic acid) and- 2 changes of absolute ethanol for 10 min each, dried and counted in a Liquifluor-toluene mixture in a Packard Tricarb liauid-scintillation counter. In some cases DNA samples were added directly to 10 ml of Aquasol (New England Nuclear) and counted (table 1).

Electron microscopy L cells were fixed, embedded, stained and examined as previously described [4]. Essentially fixation was performed in 2% OsO,-0.1 M phosphate buffer (pH 7.4) for 1 h, then in freshly prepared 10 % paraformaldehyde in the same buffer for 20 h, all at 4°C.

Source of chemicals DNase I and RNase A were purchased from Worthington Biochemicals Corp., Freehold, N.J. Ethidium bromide was a gift from Boots Pure Drug Co. Ltd,

5&Q 01

I 2

4

6

6

IO

Fig. 1. Abscissa: days; ordinate: % initial cell number. Growth of L cells in the presence of 1, 2 and 5 pg/ml EB. The cell numbers are expressed as cumulative numbers. The actual cultures were diluted with fresh medium every day or every other day, and the concentration of EB was maintained. Cells that had ceased growth were diluted only slightly to supply some fresh nutrients. Nottingham, UK. The following radioactive materials were obtained from Schwarz-Mann Bio-Research: SH-thymidine (15.6 Ci/mmole); Y-thymidine 50.8 (mCi/mmole).

RESULTS Growth patterns of L cells in the presence of ethidium bromide (EB)

The growth response of L cells treated in suspension culture with 1, 2 and 5 pg/ml EB is shown in fig. 1. In the presence of 1 or 2 pg/ml EB growth slowed down during the first 24 h of treatment and ceased after 3 days. Structural changes in mitochondria of these cells are depicted in figs 2 and 3. There was Exptl Cell Res 72

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Margit M. K. Nass

Fig, 2. Electron micrograph of control L cells. Mitochondrial profiles are filled with cristae that traverse the entire width of the organelle. x 24 000. Fig. 3. Electron micrograph of L cells grown in the presence of 1 yg/rnl EB for 3 days. Mitochondrial profiles appear enlarged, the number of cristae is reduced and the rudimentary cristae tend to be arranged in whirllike fashion. The mitochondrial matrix is less dense than in control organelles. x 24 000.

Exptl

Cell Res 72

Effects of ethidium bromide on mitochondrial DNA synthesis

a slight decline in cell viability, detected by dye exclusion tests, after longer treatment (5 days) and at higher concentrations of EB. It was shown earlier [4] that in similar experiments in the presence of 1 or 2 ,ug/ml EB the concentration of cytochromes a + a3 and b fell significantly, b at a lower rate than a i a,; cytochrome c1 7mc actually increased. Treatment with 0.1 ,ug/ml EB resulted in a more gradual inhibition of growth than treatment with higher concentrations. Cell growth resumed after ethidium bromide was removed by resuspension of the cells in fresh medium (fig. 4). There was a lag period of one to several days before growth resumed, depending on the length of exposure to the drug. A growth rate almost equal to that of control cells was finally reached in cells that were originally treated with 1 ,ug/ml EB for 2 days. A 5 day treatment still resulted in growth recovery, but the lag period was longer and final growth rate was

'60

I

600: 500400300200

-

100 -

60

-i il

50

I I

r

Fig. 5. Abscissa: fraction number; ordinate: (left) O-O 3H radioactivity (cpm); (right) O-O density (g cm-s). Mitochondrial DNA of L cells grown during logarithmic phase in the absence (upper panel) and presence of 1 pg/ml EB for 20 h. Cells were exposed to 0.2 &i/ml SH-thymidine for 3 days prior to EB treatment and continuously during treatment. Mitochondrial DNA was centrifuged to equilibrium in CsCl-EB gradients. Mitochondria were not subjected to DNase treatment. DNA II therefore represents primarily nuclear DNA. DNA I of control mitochondria consists of mitochondrial covalently closed circular DNA.

CONTROL

240

0

t

0

215

4

s

12

16

20

24

Fig. 4. Abscissa: days; ordinate: % initial cell number. Recovery of growth of L cells after a 2-day exposure to 0, 1, 2 and 5 ,ug/ml EB. At arrow, the cells were washed and resuspended in fresh medium without EB.

below that of control cells. Cells treated with 5 pug/ml EB died after 8 to 10 days. Preliminary cell cloning experiments have shown that the majority of cells treated with 2 ,ug/ml EB for 2 days were capable of forming clones on EB-free agar. The growth of BHK cells and BHK PyY cells cultured in monolayers was similarly Exptl Cell Res 72

216

Margit M. K. Nass

20

a

25

30

20

25

b

inhibited by ethidium bromide. The treated cells remained attached to the glass but grew more slowly than control cells. Studies of cells treated for longer time periods are underway to check for EB resistance. The growth curves shown in figs 1 and 2 are typical, but some variations in the degree of response have been encountered in different experiments. Treatment of mitochondria with DNase after exposure to ethidium bromide

It was consistently observed that after EB treatment of cells, in contrast to control cells, digestion of isolated mitochondria with DNase did not satisfactorily remove all contaminating nuclear DNA, even after a 60 min digestion with 50 pug/ml DNase or a 30 min digestion with 100 pg/ml DNase at room temperature. Extra care was therefore taken to keep nuclei intact during homogenization and to remove nuclei and nuclear fragments during differential centrifugation of cell homogenates. The mechanism for the apparent partial resistance of DNA to enzymatic digestion by DNase I after treatment of cells with EB is not yet clear. Exptl Cell Res 72

30

Fig. 6. Abscissa:fractionno;ordinate: (left) SH cpm x 10-e, O-O; (right) relative fluorescence x --- x : density (g cm-a). Mitochondriai DNA of BHK cells grown and treated with EB exactly as described in fig. 5. Both the radioactivity and amount of mitochondrial DNA, as measured fluorimetrically, are plotted. (a) control; (b) EB-treated.

Structural change of pre-existing closed circular mitochondrial DNA in the presence of EB

L cells, BHK cells and BHK-PyY cells were grown for 3 days in the presence of 0.2 ,&i/ml 3H-thymidine. Ethidium bromide (1 pg/ml) was then added to half of each culture and the cells grown in the presence of the isotope for 24 h. The distribution of radioactivity in mitochondrial DNA species is shown in figs 5, 6 and 7. It is apparent that the covalently closed circular DNA species (I) of EB-treated cells is no longer detectable in its normal position in CsCl-EB gradients as determined both by radioactivity (figs 5-7) and by fluorometric DNA analysis (fig. 6). The position of nicked and linear DNA (II) remained unaltered. DNA II in these experiments consisted primarily of nuclear DNA since isolated mitochondria were not digested with DNase to avoid preferential destruction of mitochondrial DNA in these organelles that possibly were membrane-damaged. The fact that even DNA labeled for 3 days prior to EB treatment was not detectable in position I (figs 5-7) demonstrates that all

Effects of ethidium bromide on mitochondrial DNA synthesis

preexisting DNA has been affected in 24 h. Preliminary experiments with L cells indicated that even after a 2- and 4-h treatment most of the DNA I was altered and only about 20 and 10 %, respectively, of the closed circular DNA fractions were still detectable in the normal position of the gradients. After reversal of EB-treated cells in EB-free medium for one week, covalently closed DNA was again apparent in its normal position. How early it reappears and by what mechanism has not yet been determined.

I

I

217

I 160

Mitochondrial DNA synthesis and decay of radioactivity in the presence of EB

To obtain a clearer picture of the fate of mitochondrial DNA during EB treatment, double-labeling experiments were performed in which mitochondria were DNase-treated to analyse total extractable mitochondrial DNA rather than the covalently closed circular species alone. Both the incorporation of 14C-thymidine during the exposure to EB and the decay of 3H radioactivity in preexisting DNA was followed. L cells were labeled with 3H-thymidine for 3 days as described above. The cells were then washed, divided into 2 cultures and resuspended in fresh medium, one culture receiving 2 ,ug/ml EB. After a 12 h period to deplete intracellular pools of 3H-thymidine and to allow EB to become fully effective, 14C-thymidine was added to label newly synthesized DNA. Cells were harvested 2 days and 4 days after the addition of EB. Fig. 8 (upper 2 panels) shows typical profiles of control mitochondrial DNA in CsCl-EB gradients. After 4 days of growth newly synthesized 14C-labeled DNA clearly exceeded the older 3H-labeled DNA. In contrast, after treatment of cells with ethidium bromide, only one broad peak was observed. Two main points are apparent: First, pre-existing CH-labeled) DNA is not lost but bands now close to the density of

7. Abscissa: fraction no; ordinate: (left) O-O *H radioactivity (cpm); (right) O-O, density(g cm-$). Mitochondrial DNA of BHK-PyY cellstreated with EB as describedin fig. 5. Fig.

nicked DNA. Second, an inhibition of the incorporation of l*C-thymidine into M-DNA occurs. The ratios of 14(J3H counts based on isotope incorporation per pg DNA (table 1) reflect this inhibition. When the counts were expressed per mg M-protein or per lo6 cells, higher counts of tritium label in pre-existing M-DNA of EB cultures as compared with control cells were observed. This may reflect the lack of cell growth and consequently a lesser degree of isotope dilution in EB-treated cells. It is also apparent (table 1) that in control cells M-DNA I showed greater 3H and 14Cthymidine incorporation than M-DNA II in Exprl

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218

Margit M. K. Nass

Table 1. Incorporation of 14C-thymidine and decay of 3H-thymidine in mitochondrial and nuclear DNA during long-term treatment with EB

cpm/,% DNA

cpm/mg M-prot.

cpm/lO’ cells

lQJ3H

1%

3H

14C

C M 1’ C M II CN

1.21 1.11 1.49

762 448 -

529 403 -

11.3 6.1

2 2 2

EB M I EB M II EB N

0.70 0.60 1.53

503 565 -

717 946 -

5.5 6.1

7.8 10.3 -

4 4 4

CM1 C M II CN

6.44 4.74 8.69

391 304 -

2 -

3.4 2.6

0.5 0.5

4 4 4

EB M I EB M II EBN

0.99 0.85 1.96

133 259 -

138 253 -

Days of treatment

DNA

2 2 2

-

-

1.2 2.3

3H

-

-

-

9.3 6.0

1.4 2.5

a Abbreviations: C, control; EB, ethidium bromide (2 pg/ml); M I, M II, mitochondrial DNA; N, nuclear DNA. Experiment similar to that described in fig. 8. DNA I and II from EB samples are analogous to fr. 2%39,40-55 (2 days), fr. 35-40, 41-52 (4 days).

contrast to the reverse trend in EB-treated cells. It should be noted, however, that the essentially single peak of M-DNA from EBtreated cells was arbitrarily divided into DNA I and DNA II fractions corresponding to the buoyant density ranges of control DNA I and II. These fractions therefore do not necessarily contain functionally and structurally comparable DNA species. The DNA (fig. 8) was also examined electron microscopically by the DNA monolayer technique as described [6]. DNA I of control cultures consisted entirely of circular DNA; DNA II consisted of 5&70 % nicked circular DNA and 50-30 y0 linear DNA. The single DNA peak of EB-treated cultures consisted almost entirely of linear DNA. There were approx. 5 % circular DNA molecules in DNA I and none in DNA II after 2 days of EB treatment, and none were detectable after 4 days. The circular DNA molecules were covalently closed as deterExptl

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mined by band sedimentation in CsCl at pH 12.6. The few circular molecules seen after EB treatment contained approximately the same relative proportion of DNA monomers, dimers and oligomers as control cells. Similar studies are in progress with mitochondria of BHK and BHK-PyY cells. Preliminary studies have shown that EBinduced ultrastructural changes were less marked in mitochondria of BHK and BHKPyY cells than of L cells. Nuclear DNA synthesis in the presence of ethidium bromide Tables 1 and 2 indicate that nuclear DNA synthesis is not inhibited by EB in L cells, BHK and BHK-PyY cells under the conditions tested. There is a trend that the specific activity is actually greater in EB-treated cells than in control cells. This effect is more pronounced after brief treatment (1 day) than

Effects of ethidium bromide on mitochondrial DNA synthesis 300

I

I C 2 DAYS ! ’

EB 2DAYS

I

I

I

I

C 4 DAYS

; 4:

1

I

P: 9:

EB 4 DAYS

-

Fig. 8. y$)j~---~,

c

100 0It

219

_

A 30

40

after prolonged treatment with EB, e.g. longer than 2 days (table 1). In the latter case nuclear DNA synthesis is to some degree inhibited, although not as greatly as mitochondrial DNA synthesis. The reason may be that after prolonged treatment with EB the effects of this drug on cell functions are undoubtedly much less specific than after shorter treatments, and in this case cell viability is affected as well. Yields of mitochondrial DNA after EBtreatment Table 3 shows that the amount of M-DNA recovered per mg M-protein does not change very significantly during 2 days of treatment with EB. A slightly lower yield was obtained after 4 days of treatment. If the yield of M-DNA is related to unit number of cells, it is also slightly lower in cells treated with EB for 4 days than in control cells. The lower

50

4l--

Abscissa:

fraction no; ordinate: SH; O-O lpC radioactivity

. Mitochondrial DNA of L-cells centrifuged to equilibrium in CsCl-EB gradients, showing the incorporation of %-thymidine during a 2-day and 4-day treatment with 2 fig/ml EB and the decay of 3Hthymidine incorporated into DNA prior to EB treatment. DNA was isolated from approximately equal amounts of M-protein. Details stated in text.

yield based on concentration of mitochondrial protein partly reflects the consistently observed greater protein content in mitochondrial fractions of EB-treated cells (table 4). The exact reason for the latter observation is not yet clear. DISCUSSION These results show that the synthesis of all species of L cell mitochondrial DNA, as indicated by incorporation of 3H-thymidine, is selectively inhibited and that nuclear DNA synthesis is unaffected or actually stimulated. The stimulation of nuclear DNA synthesis has been observed in all three cell lines studied. Whether this apparent stimulation represents increased DNA synthesis or repair is not yet known. If it is increased synthesis it may be triggered by an imbalance in nuclear-mitochondrial interaction as a Exptl

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220

Margit M. K. Nass

Table 2. Specific activities of nuclear DNA

Table 4. Yield of protein in mitochondrial

from control and EB-treated L cells, BHK cells and BHK-Py Y (polyoma virus transformed) cells

fractions from control (1 or 2 pug ml/EB)

Cells

Treatment

L

Control, 2 days EB (1 pup/ml), 2 days Control, 2 days EB (2 pg/ml), 2 days aControl, 1 day aEB (0.1 ,ug/ml), 1 day ‘EB (1 .O ,ug/ml), 1 day Control, 2 days EB (2,4d), 2 days

BHK BHK-PyY

BHK-PyY

2 862 4500 4 850 5 662 740 848 1 795 3 190 5 199

n ‘H-thymidine (0.2 @/ml) was added 15 min after the addition of EB. In all other cases listed sHthymidine was added 24 h prior to and during EB treatment.

result of EB-induced mitochondrial damage [4], which would limit the supplies of ATP and other products vital to cell function. Initially, nuclear activity may increase and lead to an increased synthesis of mitoTable 3. Yields of DNA from DNase-treated mitochondria of control and EB-treated cells Experimental condition

pg DNA/mg protein

L cells Control, 2 days EB (2 @g/ml) 2 days Control, 4 days EB (2 ,ug/ml) 4 days

1.76 1.51 1.17 0.78

BHK cells Control, 2 days EB (2 pg/rnl) 2 days Control, 4 days EB (2 pg/ml) 4 days

2.98 2.60 2.84 (2 603)” 1.89 (1 832)”

mito

The DNA values represent total extractable mitochondrial DNA recovered from regions I and II of CsCl-EB gradients. After removal of the dye and dialysis, DNA was determined spectrophotometrically a cpm/,ug DNA. *H-thymidine was given to cells 24 h prior to and during EB treatment. Exptl Cell Res 72

and EB-treated

Experimental condition

Days

pg mito. protein/lo6

L cells (1) Control EB (2) Control EB (3) Control EB

2 2 2 2 2 2

7.8 10.7 10.7 16.3 9.8 12.7

BHK cells (4) Control EB (5) Control EB Control EB

2 2 2 2 4 4

7.7 11.9 11.3 13.3 10.7 16.7

BHK-PyY cells (6) Control EB

;

2.8 5.2

cells

cells

chondrial proteins which are under the control of nuclear genes. In agreement, we have shown an increase in the concentration of cytochromes c1 and c during EB treatment [4]. Whether the increased yield of protein in mitochondrial fractions observed here is similarly related to the activity of nuclear genes or merely due to increased contamination by cytoplasmic protein cannot yet be resolved. Alternatively, the higher specific activity of nuclear DNA may be a consequence of the differential and/or altered permeability or affinity of nuclear and mitochondrial membranes to EB. The results also show that the structure of covalently closed mitochondrial DNA is altered as a consequence of exposure to EB. The effect of EB was manifested as a displacement of DNA I in CsCl-EB gradients. The structural change involved essentially all preexisting DNA as well as the small amounts

Effects of ethidium bromide on mitochondrial DNA synthesis

of DNA that appeared to be newly synthesized under partially inhibiting conditions. The displacement of covalently closed DNA I may at least partly be due to a change to greater superhelix density of M-DNA, an event which requires nicking-closing cycles during EB treatment [ll]. In addition, the nicking and breakage of DNA I appears to be in excess to any closing that may take place. This is apparent from electron microscopic analysis and band centrifugation in alkali of the M-DNA, showing a much greater percentage of linear M-DNA and less circular DNA in EB-treated cells as compared with controls. A factor of uncertainty, however, which is difficult to eliminate in experiments involving EB treatment of cells is the exact effect of DNase digestion during isolation of mitochondria. Both the degree of structural integrity of mitochondrial DNA and the possible presence of residual small pieces of nuclear DNA must be considered. Details on the structural changes of mitochondrial DNA of EB-treated cells will be reported elsewhere. It is apparent that mitochondrial DNA, regardless of its structure, is retained although perhaps slightly diminished during at least 4 days of treatment with EB, a period corresponding to four generations of control cells. This situation is clearly different from that in yeast where parental mitochondrial DNA may become rapidly degraded during EB treatment [13, 141. However, in the L cells electron micrographs of EB-treated cells revealed a considerable structural heterogeneity in the mitochondrial population from cell to cell and within a cell [4]. Since the isolated mitochondria reflect the average situation, it is obviously not possible to determine whether the observed effects are representative of events taking place in all mitochondria and all cells, or whether a small

221

fraction of mitochondria and cells may become non-viable mutants. The use of ethidium bromide in animal cells, especially in combination with other known selective inhibitors of macromolecular synthesis, promises to become an effective tool for studying processes of mitochondriogenesis that are under nuclear and/or mitochondrial control. As part of the John RunnstrGm Memorial Issue of Experimental Cell Research, this paper is dedicated to Professor John RunnstrSm whose keen interest was encouraging when we discovered DNA in mitochondria (refs [24-261) at a time when this concept was still unacceptable to the majority of scientists. The author is grateful for the skilled technical assistanceof Mrs-M. Buurma, A. Hathaway, D. Reinhardt. H. Williams and A. Trischitta. This w&k was supported by grant POl-AI07005 and Career Development Award K03-AI08830 from NIH. Portions of the results were presented at the Cell Biology Meetings in San Diego, November 1970, and the CNRS Meeting in Port-Cros, France, May 1971.

REFERENCES 1. Slonimski, P P, Perrodin, G & Croft, J H, Biochem bioohvs res commun 30 (1968) 232. 2. Perlman, P s & Mahler, H R, B&he& biophys res commun 44 (1971) 261. 3. Kellerman, G M, Biggs, D R & Linnane, A W, J cell biol 42 (1969) 378. 4. Soslau, G & Nass, M M K, J cell biol 51 (1971) 514. 5. Nass, M M K. In preparation. 6. - Proc natl acad sci US 67 (1970) 1926. 7. - J cell biol 47 (1970) 147~. 8. Horwitz, H B & Holt, C E, J cell biol 49 (1971) 546. 9. Radsak. K. Kato. K. Sato. N & Koorowski. H. Exptl c&l ies 66 (i97i) 410: * ’ ’ 10. Riou. G & Delain. G. Proc natl acad sci US 64 (196<) 618. ’ ’ 11. Smith, C A, Jordan, J M & Vinograd, J, J mol biol 59 (1971) 255. 12. Meyer, k R’& Simpson, M V, J biol them 245 (1970) 3426. n 13. Goldring, E S, Grossman, L I, Krupnick, D, F2Z D R & Marmur, J, J mol biol 52 (1970) 14. Per&n. 231 (197i) 15. Goldrina. J bacter&l

P S & Mahler. . H R. , Nature new biol 12. E S. Grossman. L I & Marmur. I J.I 107 11971) 377. ’ Exptl

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Margit M. K. Nass

16. Nagley, P & Linnane, A W, Biochem biophys res commun 39 (1970) 989. 17. Zylber, E, Vesco, C & Penman, S, J mol biol 44 (1969) 195. 18. Knight, E Jr, Biochemistry 8 (1969) 5089. 19. Perlman, S & Penman, S, Biochem biophys res commun 40 (1970) 941. 20. Azzi, A & Santato, M, Biochem biophys res commun 44 (1971) 211.

Exptl Cell Res 72

21. 22. 23. 24.

Nass, M M K, J mol bio142 (1969) 521. - Ibid 42 (1969) 529. - Proc natl acad sci US 56 (1966) 1215. Nass, M M K & Nass, S, Exptl cell res 26 (1962) 424. 25. - J cell biol 19 (1963) 593. 26. Nass, S E & Nass, M M K, J cell biol Ibid 19 (1963) 613.