DNA polymerase γ, cytochrome c oxidase and mitochondrial integrity in rabbit spleen lymphocytes stimulated with concanavalin A

Experimental

DNA

POLYMERASE

MITOCHONDRIAL

Cell Research 127 (1980) 269-216

y, CYTOCHROME

INTEGRITY

IN RABBIT

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WlTH L.

VANHEU1.E.’

SPLEEN

LYMPHOCYTES

CONCANAVALIN G.

VII.LANI’

AND

A x and

S. SI’:\DARI’

SUMMARY The activities of two mitochondrial enzymes: DF\;A polymerase y and cytochrome c oxidasc. have been compared with the kinetics of nuclear DNA synthesis and levcis of the nuclear DN.4 polymerases a and /3 in rabbit spleen lymphocytes untreated or stimulated with concanavalin A (ConA). At early initiation of the culture, before the maximum increase of both the replicative a-polymerase and nuclear DNA synthesis, a simultaneous enhancement in the activities of the two mitochondrial enzymes was observed, just preceding and paralleling the expansion of the ATP pool. It is suggested that this is a reflection of increased mitochondrial activity providing the energy which results in an elevation of the concentration of dNTPs to values which are optimal for the replicative u-polymerase. During the later stages of incubation of lymphocytes. the activities of both DNA polymerase y and cytochrome c oxidase decrease. paralleling a progressive loss of integrity of the mitochondrial structures within an increasing proportion of the lymphocyte popuiation.

Mammalian cells contain three distinguishable polymerases (cu, p, y). Although the direct assignment of their functions in DNA replication and repair is hampered by the lack of conditional mutants defective in DNA synthesis, the conclusions emerging from recent studies are as follows: DNA polymerase u is mainly responsible for DNA replication in nuclei [l-5]. DNA polymerase p is able to repair UV or chemically damaged DNA in nuclei [4, 51, DNA polymerase y replicates mitochondrial DNA (mitDNA) [4, 51. Previous work [6], in which we have studied the correlation between the activities of DNA polymerase U, /3 and y and the replicating DNA in spleen lymphocytes stimulated by different doses of concana-

valin A (ConA). indicated that the activities of DNA polymerase y fell rapidly in both untreated and stimulated lymphocytes wi?hin the first 20-30 h of incubation in culture. The recent finding that DNA polymerase y is the only polymerase present in mitochondria [7-91 and that it replicates mitDNA [4. 51 prompted us to examine the activity of another mitochondrial enzyme. cytochromc c oxidase, in untreated and ConA-stimulated lymphocytes. In this work we will show that the activities of both DNA polymerase y and cytochrome c oxidase show similar changes during the culturing of lymphocytes. Both * Present address: ford University 94305, USA.

Department School of

of Hiochemistrv. Medicine. Stanford.

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Hardt et al. cubating the cells in 0.1% trypan blue for 2-3 min at room temperature. The percentage of stained cells was determined as the mean of duplicate counts of 400 cells. Viability was also evaluated by analysis of the integrity of the cytoplasmic structures by electron microscopy.

Electron

1. Ordinate: (lefr) cytochrome c oxidase (*) and DNA polymerase y (0) activities, expressed as percentages of the values at start; (right) percentage of cells labelled with PHlTdR after a nulse of 30 min

Fig.

(m-0).

-

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The variations of cytochrome c oxidase and DNA polymerase y activities during short-term lymphocytes cultures, (a) stimulated by 5 pg/ml ConA or (b) unstimulated, are correlated with the nuclear DNA synthesis rate reported as percentage of [3H]TdR pulse-labelled cells. 100 % of the activity for cytochrome c oxidase=8.8 mEq/mg proteins; for DNA polymerase y=O.3 U/mg proteins.

enzyme activities increase during the first few hours in culture and then decrease just before the onset of both the replicative a-polymerase enzyme and the nuclear DNA synthesis. A subsequent decline in the activities of the two mitochondrial enzymes can be correlated with the progressive appearance in the cultures of cells showing fewer or damaged mitochondrial structures and of cellular death by oedema [ 10, 111. MATERIAL Animals

AND

METHODS

and culture techniques

Spleens were aseptically removed from young adult rabbits and gently teased on a stainless nylon sieve (60 mesh). Cells were recovered in PBS, centrifuged at 700 g for 10 min, washed and centrifuged again and resuspended at a concentration of 5~10~ cells/ ml in minimum Eagle’s medium (Gibco) supplemented with non-essential amino acids, and 5% decomplemented fetal calf serum (FCS) Rehatuin. One ml cultures were distributed in plastic tubes (Falcon plastics). Incubation was carried out at 37°C in an air+ 5% CO, mixture. When used. ConA (Miles) was added at the beginning of the culture to .a final concentration of 5 pg/ml. Viability was evaluated by trypan blue dye exclusion in control cultures by inExp Ceil Res 127(1980)

microscopy

Spleen fragments were taken for electron microscopic examination immediately after killing the rabbits. Spleen cells were taken after the filtration recovery step and centrifugation steps referred as above and during the course of the culture at the times indicated in the text. Samples were dipped in cacodylatebuffered glutaraldehvde and post-fixed in osmic acid or directli fixed in bsmic acid, contrasted in uranyl acetate and embedded in Epon-Spurr mixture (equal volumes). Diamond cut ultrathin sections were examined in a Siemens 102 microscope. Glass cut 1 w sections were collected on microscope slides, stained with safranine 1% after hydrogen peroxide treatment to dispose of osmium, and photographed with a green filter on a Reichert Zetopan microscope (periplan 100X objective, 10X ocular). About 1000 cells were counted per item.

Incorporation of r3H]TdR autoradiography

and

Tritiated thymidine (10 &i/ml, 5 Ci/mM Amersham) was added to the cultures 30 min prior to the harvesting of the cells, at the times indicated in figs 1 and 2. The harvested cell suspensions were washed in PBS, incubated for 10 min at room temperature in a mixture 50% PBS/SO% fixator (3 vol methanol and 1 vol acetic acid) and then for 20 min at 4°C in the same fixator. The cells were then spread on dry slides, air-dried, washed and prepared for autoradiography as previously described [6]. They were then stained by the UNNA method. The percentage of labelled cells was estimated by counting two samples of lo3 cells for each culture.

Preparation of cell extracts, cell counts and protein content The cells of 10 culture tubes were pooled and extensively washed. Before the last centrifugation the number of cells was determined with a hematimeter Thomas; no significant change in the number of cells was observed during the course of the culture. The cells were then homogenized in 1 ml of 10 mM Tris buffer (pH 7.5), ti mM KCl, 1.5 mM MgCl,, 0.5 mM DTT in a glass homogenizer. The homogenate was then made 0.1 M KCl, 0.1 M KH,PO, (pH 7.2), 0.5% Triton X-100 and sonicated 2x5 set with the microtip of a Branson Sonifer at a setting of 50 W. This extract was used as an enzyme source both for assays of DNA polymerases o(, /3 and y and of cytochrome c oxidase. The protein content of the extracts was determined by the method of Lowry [12], and remained fairly constant throughout the course of the culture.

Tris buffer (pH 8.5). 50 mM KH,PO,, 0.13 M KCI. 0.5 mM MnCI,. I mM DTT. 250 pg/mi HSA. ?O figi ml poly(rA)-olieo(d.f),, I% and 50 PM [:‘H]TTl’ ( 1 S(H) cpmlpmol). Under these conditions the assay wa5 strictly specific for the y-polymerase since P-polymerase activity on poly(rA)-olipo(d.f),,_,, ic inhibited by phosphate and u-polymerasc does not utilirc Ihi\ po1ynucleotide as template primer [3]. .4 unit is defined as I nmol of total deoxynucicotide incorporation into acid-insoluble form in 60 min at 37’C. The template\ were prcparcd a pveviouhl! described 161. oer”!

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I.‘iu. 2. Ortlirlcr/c~: C/c/i) A’I‘I’ pool (:?). DNA polymerase y (Cl and DN.4 polymerasc (I (0) activities cxpressed as percentages of the value at start: (,-i#h() percentage of ceils labellcd with [:‘H]T~K after a pulse of 30 min (O--O). Irisrr (h ). Ab.scisw: lime (hours): or-dincrre: ISA polymerasc p activitv expressed as a percentage of the value at 5tart in t . ConA-stimulated: n , control cultures. The variation\ of DS.4 polymerase u and y activities and of the ATP pool during short-term lymphocytes cultures. ((0 stimulated by 5 pg/ml Con.4 or (h) unstimulated. are correlated with the nuclear DNA synthesis rate rcportcd as percentage of i:‘l-i I’l‘dK pulse-labelled ceils. 100% of the activity foi DNA polymerasc 0~ I.2 U/mg proteins; for DhA polymerase p=O.7 U/mg proteins: for DNA polymcra\e y=2.1 L!mg proteins: value at start of the ATP pool=532 pmolil0” cells. Similar results were obtained by culturing the cells with IS g>l aphidicolin (no nuclei labelled).

A.ssuy (?i’c.vtoc.ll,.ol?lc

Cytochromc c. oxidase activit! wax a\saqed by the method of Cooperstein & Lazarow [ 141 and expresed in the units dcfincd by these authors. ‘l‘he result\ arc the mean of duplicate experiments on two dill‘erent concentration\ of cell extMct\.

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polymcrtrsc

CY,/3 trntl y

Each reaction was carried out at 36°C and samples wel-L‘ taken at 6. I2 and IX min for LY- and P-polymerase. and at 3. 6 and 9 min for y-polymerase. The reaction kinetics were found to be linear during this period. .4\ previously described [6]. the u-polymerasc reaction was carried out in 20 mM KH,Po, (pH 7.2). 0.1 mM EDTA. 0.5 mM DTT. IO mM MgCI,. 250 &ml of ISA. 200 pg/ml of activated calf thymus DN.4 and all four dcoxyribonucleoside triphosphatcs at IO0 PM each with [“H]T’I‘P (1 000 cpmlpmol). Under these conditions, P-polymerasc responds with a 30%. efficiency to the n-polymerasc assay [6]. All tr-polymerase data presented hcrc have been corrected for the contribution of the /&enzyme. On the othcl hand, y-polymcrasc is ineffective under these conditions [l3]. DNA polymerase fi was assayed in 50 mM Tris buffer (pH X.5), 0.1 M KCI, IO mM MgCI,. I mM DTI‘, 250 &ml BSA. 200 &ml of activated calf thymus DNA and all four deoxyribonucleosidcs triphosphates at lo0 pM each with [:‘H]‘l’TP (I 000 cpmi pmol). DNA polymerase Q was inactivated by preincubation with IO mM NEM at 0°C for 30 min. DNA polymerase -y was assayed according to Knopf et a1. [13]. The reaction was carried out in 50 mM

o/‘tllc~ A7’P pool

I-roren lymphocytes (IO” cells) were extracted with 200 ~1 of ice-cold 5!k ‘I’CA at 4’C for 30 min under constant vortexing. r\ftcr centrifuugation. the SU~CI-natant was diluted to 400 PLI with H,O and extracted five time$ with ether to remove TC.4. Fifty @I of this solution was neutralized by addition of IO ~1 of 13.5 mM IIEPES (pH 7.5). Fifty PI of L.umit PM (I.umac Systems AG. Basel) were then added and the light emission was measured in a scintillator counter. Variability of the valuc5 obtained was approx. 10%.

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Aphidicolin 60 mM was dissolved in DMSO. This solution was diluted in culture medium to obtain a final concentration of I5 PM. When used. aphidicolin was added at initiation of the culture both in Con.4 stimulated and untreated cells.

KESULTS

Figs 1 and 2 show the results of typical experiments in which we have determined the activities of DNA polymerases in ((1) ConA-stimulated and (0) untreated spleen lymphocytes. A slight increase ill activity of y-polymerase was observed in stimulated lymphocytes, while no change was found in

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Hardt et al.

unstimulated cells, within the first lo-20 h in culture (fig. 1). These effects occur prior to the previously reported decline in activity of y-polymerase [6]. A similar increase in activity of cytochrome c oxidase was also observed in stimulated cells, whereas untreated lymphocytes showed only a small increase or, in some experimerits, no increase in cytochrome c oxidase activity. At later times of incubation the level of cytochrome c oxidase activity decreased in both stimulated and untreated cultures. These experiments were repeated several times with similar results. The only variability observed was in the timing of induction (6-12 h). The simultaneous activation of both DNA polymerase y and of cytochrome c oxidase reflects an increased mitochondrial activity leading to an expansion of the ATP pool (fig. 2). This occurs prior to both the maximum activity of the replicative a-polymerase enzyme and the onset of nuclear DNA synthesis, which grow in parallel and reach a maximum value after 48 h in culture (fig. 2). DNA polymerase p activity (inset of fig. 2B) levels off during the culture period [6]. As this enzyme is of importance in DNA repair [ 151, it may be expected that repair occurs equally in both proliferating and non-proliferating cells. The determinations of (Y-, p- and y-polymerases activities and of the ATP pool were also performed on ConA-stimulated lymphocytes grown in the presence of 15 PM aphidicolin-a specific inhibitor of DNA polymerase 01 and of DNA synthesis in mammalian cells [ 16, 171: approx. 97% of the DNA synthesis is inhibited with no effect on cell viability [18]. The rabbit lymphocytes kept synthesizing all three polymerases (a, j3, y) and ATP at the same level as in fig. 2. The results are identical and are therefore not shown. Autoradiographs Exp

Cell

Res 127(1980)

showed no detectable grains in the nuclei, against about 100 grains in the labelled nuclei of cultures grown in the absence of aphidicolin (when the autoradiographs were developed after 8 days of exposure). The cell viability and the blastic transformation was not modified. These results are of particular interest because they could be correlated with the resistance both in vivo and in vitro [16, 171, of the mitochondrial y-polymerase to aphidicolin and could as well explain why cells, in the presence of this drug, show an unchanged or even higher pool of dNTPs [ 181. Correlation between the levels of enzymes active in mitochondria and the integrity of lymphocytes and mitochondrial structures The similarities between the behaviour of DNA polymerase y and cytochrome c oxidase in cultures of lymphocytes lead us to examine the integrity of lymphocytes and of mitochondrial structures in cell cultures and to compare them with cellular types present in cross sections of spleen fragments of normal rabbits obtained immediately after killing the animals, and in lymphocyte suspensions recovered after teasing the spleen and filtering the cells through a nylon sieve (60 mesh) prior to any washing. The lymphocytes recovered by teasing and filtration are relatively homogeneous in size (fig. 3). Electron microscopic analysis reveals most of them to have well developed mitochondria with clearly visualized cristae. Some cells contain dense bodies (not shown) or round clear vacuoles (fig. 3b). Two minority classes of abnormal cells, apparently lacking mitochondria, were observed immediately after the death of the rabbit. The first class (fig. 3 a, less than 2 % of the cells) had a cytoplasm full of dense

Mitochondrial

activity

.he spleen and filtration. (a) The cell a cytoplasm full of dense bodies (arrows) and very few mitochondria. This cellular type represents about lm -CA,.- r-.^l ----..-L c--11- ,I / $1 ..11.~-.I~ 1 1

in short-term

lymphocyte

cultures

273

(N, nuclei are rather well preserved). Bar, IO00 nm. x11000.

ExpCdl Kes127 (lY80)

274

Hardt et al.

Fig. 4. Cross-section of a spleen fragment showing the presence of a ‘degenerating’ cell (D) in situ, together with intact cells. Bar, 1000 nm. x7000. Exp Cell Res 12711980)

Fig. 5. Cells cultured for 96 h. I, Intact cell, 2, degenerating cell with a pyknotic nucleus. Inset, detail of an intact cell. Bar, 1000 nm. x7000; inset x36 000.

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60

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HWE. Fig. 6. Integrity of the cytoplasmic structures of the Ivmnhocvtes cultured with 0. ConA: or A. none. koknts were performed on safraninc-stained I pm glass-cut sections. Cell viability (A-A) in control culture as determined by trypan blue dye exclusion. The total number of cells (dead 1 alive) remained fairly constant throughout the course of the culture. a\ dia the protein content of the cell pellets. Cellular harvest. treatment and counts mere as described in Material and Method\.

bodies, while the second class (fig. 5. 2% at the moment of animal death, later increasing slightly) showed a cytoplasm riddled with vacuoles and a nucleus smaller than in the other cells. Presumably these two cell types are dying: the first class after very active phagocytosis [ 111, the second class by cellular ocdema and cytoplasmic disorganization [IO, I I]. These degenerating cellular types do not seem to be due to the handling of the lymphocytes since they are also observable in cross sections of spleen fragments of normal rabbits obtained immediately after killing the animals (fig. 4). In culturing lymphocytes, most of the cells maintain the integrity of their cytoplasm during the first 24 h, after which cells of the second minority class, riddled with vacuoles and containing fewer or damaged mitochondrial structures, appear progressively more and more frequently (figs 5. 6), principally in unstimulatcd cultures.

ConA preserves the cytoplasmic structures. At very early times in culture. the number of dead cells, measured by trypan biuc staining. is higher than the number of cells which have already lost their integrity in viva, as revealed by electron microscopy. 20% against 2’3 (fig. 6). The staining probably reflects changes in membrane semipermeability due in part to handling of the cells prior to culturing (increasing the vclocity of centrifugation during washing t-esuits in an increased number of stained cells) and does not necessarily corrciatc with the loss of organization of organelies or of enzyme activity [I I]. After 48 h in culture. the data of the two tests arc similar. DlSCUSSlON The purpose of this work was to e!uc&tc the biological significance of the decline 01 the DNA polymerase y activity in cultures of both ConA-stimulated and untreated lymphocytes [6]. Assays of the y-polymerase activity at earlier times in culture revealed that this decline in the activity ot the enzyme is preceded by an increase in activity or a stabilisation of activity in ConA-stimulated and untreated iymphocytes, respectively. Similar behaviour was observed for the mitochondria! enzyme. cytochrome c oxidasc. The electron microscopic analysis of cultured lymphoc::tes revealed that most of the cells maintain their integrity during the first 24 h in culture. We have shown that two enzymes of the mitochondrial metabolism arc functionally active during this period. which precedes the maximum increase of both the activity of the replicativc tr-polymerase enzyme and the nuclear DNA synthesis. At later times in culture, more cells with disorganized cytoplasm and damaged mitochondrial structures were seen and a similar

276

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et ul.

decline in the activities of y-polymerasc and of cytochrome c oxidase was also observed. The increase in level of activity of DNA polymerase y and cytochrome (’ oxidase at early initiation of the lymphocyte cultures would be in agreement with the analogous increase in y-polymerase activity which we observed at early times of the S phase in synchronized HeLa cells [ 191 prior to maximum activation of the replicative cu-polymerase and the onset of nuclear DNA synthesis. .Qt that time, we were not aware that y-polymerase was the mitochondrial DNA polymerase and no efforts were made to obtain nuclear or cytoplasmic fractions free of mitochondria. DNA polymerase y has K,, values for deoxyribonucleoside triphosphates which are at least one order of magnitude lower than those of the replicative cY-polymerase [20], leading one to expect that y-polymerase should function more efficiently than a-polymerase at the beginning of the S phase when the concentrations of deoxyribonucleotides (dNTPs) are still relatively low [21, 221. We believe that the increases in DNA polymerase y, cytochrome c’ oxidase and pool of ATP observed here at early times after ConA stimulation are consistent with increasing mitochondrial activity. Reasoning by analogy with HeLa cells, hamster cells and phytohemagglutinin-treated lymphocytes [21, 22, 231 where it is known that the rate of DNA synthesis increases simultaneously with the expansion of dNTPs pool, one could suggest that these active mitochondria provide the energy which results in an elevation of the concentrations of dNTPs to values which are optimal for cY-polymerase [20]. DNA polymerase (Y is then responsible [6] for the nuclear DNA synthesis associated with the ConA stimulation in lymphocytes. Erp C’r//Kes I2 7 !IY&O)

We are indebted to R. I,egas for expert technical assistance and to M. Goldfinger and Dr M. Decrolv for friendly initiation to the method of measurement of cytochrome c oxidase activity. ATP analvsis was perf&med by Dr J. Kriippa. Thanks are aIs& due to Dr Sels for helpful discussions. This investigation was supported by the research contract Euratom (U LB no. 224-76- 1 BlO B).

REFERENCES Bollum, F J. Prog nucl acid res mol hiol I5 (1975) 109. Weissbach, A, Ann rev biochem 45 (1977) 25. Falaschi, A d Spadari, S, PNA synthesis: Present and future (ed 1 Molineux & 41 Kohiyama) p. 487. Plenum Press, New York (1978). Htibscher, U, Kuenlle, C C, I.imacher, W, Scherrer. P & Spadari, S. Cold Spring Harbor symp quant biol 1 (197X) 62.5. 5. Hiibschcr, U, Kuenzlc, C C & Spadari, S, Proc natl acad sci US 76 (1979) 23 16. 6. Spadari, S, Villani, G dt Hardt. N. Exp cell res 113 (1978) 57. 7. Boldcn. A, Pedrali-Noy, G & Weissbach, A, J biol chcm 252 (1977) 335 1. 8. Hiibscher, U, Kuenzle, C C & Spadari, S, Eur j biochem 81 (1977) 249. 9. Bertazzoni. U, Scovassi, A I & Brun, G, Eur j biochem 81 (1977) 237. 10. Majno, G, La Gattula. IM & Thompson, T E. Virchows arch pathol anat physiol 333 (1960) 421. II. Bessis, M. Ciba symposium on cellular injury (ed A V S De Reuck &J Knight) p. 287. Little-Brown & Co., Boston (1964). 12. Lowry, 0 H. Roscbrough, N J, Farr, A L 8r Randall, R J, J biol them 193 (1951) 265. 13. Knopf. K W, Yamada, .M & Weissbach, A, Biochemistry 15 (1976) 4540. 14. Cooperstein, S J & Lazarow, A, J biol chcm 189 (1951) 665. 1.5. Waser, J, Hiibscher, U, Kuenzle, C B Spadari, S, Eur j biochem 97 (1979) 368. 16. Ikegami. S, Taguchi, T, Ohashi, M, Oguro, M, Nagano. H & Mano. Y, Nature 275 (1978) 458. 17. Pedrali-Noy, G & Spadari, S, Biochem biophys res commun 88 (1979) 1194. 18. Pedraly-Nay, G, Spadari, S, Miller-Faures, A, Miller, A 0 A. Kruppa, J & Koch, G, Nucleic acid res 8 (1980) 377. 19. Spadart, S & Wetssbach. A, J mol biol X6 (1974) II. 20. - J biol them 249 (1974) 5809. 21. Skoog, K L. Nordenskjtild, B A & Bjursell, K G, Eur j biochem 33 (1973) 428. 22. Bray, G Sr Brent, 1‘ P, Hiochim biophys acta 269 (1972) 184. 23. Tyrsted, G, Exp cell rcs 91 (1975) 429. Received March 13, 1979 Revised version received December 7, 1979 Accepted December IY, 1979