Cytochrome c oxidase subunit II mRNA levels during T-lymphocyte proliferation and liver regeneration

Biochimica et Biophysica Acta, 1092 (1991) 184-187 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100141Y

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Cytochrome c oxidase subunit II mRNA levels during T-lymphocyte proliferation and liver regeneration A l e j a n d r o Otero, Xosd R. Bustelo a n d J a i m e G 6 m e z - M f i r q u e z Departamento de Bioqulmicay Biolog[a Molecular, Facultad de Biologia, Unioersidadde Santiago de Compostela, Santiago de Compastela, Galicia (Spain)

(Received 18 September 1990)

Key words: Cytochrome c oxidase; Hepatectomy; Cell proliferation; (Rat T-lymphocyte)

We studied here the variations in the mRNA levels of the mitochondrially-encoded subunit !1 of cytochrome c oxidase (COIl) during the proliferation of thymocytes, splenic T-cells and hepatocytes. The COIl mRNA levels increased during thymocyte proliferation and decreased when they were growth arrested. However, its levels remained nearly constant during splenic T-cell proliferation and liver regeneration after partial hepatectomy. The different pattern of COIl gene expression in the cellular systems analyzed suggests that an increment in the oxidative metabolism could not always be necessary during cell proliferation.

The mammalian cytochrome c oxidase (EC 1.9.3.1) is a mitochondrial multisubunit enzyme which catalyzes the aerobic oxidation of cytochrome c with reduction of 02 to H20 and formation of a transmembrane protonmotive force'. COIl is one of three catalytic subunits encoded by the mitochondrial genome [1] and provides the locus for the binding of one copper atom and cytochrome c [2]. In a previous work we have shown that the steadystate levels of COII mRNA changed during the rat T-cell development [3]. Such variations in the abundance of COIl mRNA could be explained by the cellular proliferation occurring during the maturation of T-cells [3]. In the present report we analyze the relationship between the COII mRNA levels and cell proliferation in three different systems: thymocytes, spleen Tcells and liver regeneration after partial hepatectomy. In these experiments, we used as controls of cell proliferation the measurement of [3H]thymidine uptake as well as the pattern of expression of the S-phase marker historic H3 (H3) gene (reviewed in Ref. 4).

Abbreviations: COIl, ¢ytochrome c oxidase subunit I!; H3, histone H3; ConA, concanavalin A; rIL-2, human recombination interleukin 2; dNTPs, deoxynucleoside triphosphates. Corresl~ndence: J. G6mez-M~kqucz, Departamento de Bioqulmica y Biologia Molecular, Facultad de Biologia, Universidad de Santiago, Santiago de Compostela, Galicia, Spain.

Thymocytes were obtained from 2-month-old Sprague-Dawley rats and cultured ((2-4). 106 cells/ml) at 37°C in a 5% CO2 humidified atmosphere in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 10 mM Hepes (pH 7.4), 100/~g/ml streptomycin, 100 units/ml penicillin. Cells were stimulated with 6 /tg/ml of concanavalin A (ComA) plus 10 units/ml of human recombinant interleukin 2 (rlL-2). After mitogenic stimulation, total RNA was prepared and aliquots of 10 ~tg electrophoresed on a 1.5% agarose/formaldehyde gel and blotted onto nitrocellulose. Filters were sequentially hybridized with (2-4). 106 cpm/ml of the nick-translated pIL-7 plasmid, which contains a rat COII eDNA clone [5], and with a 2.5 kb EcoRI/HindIIl fragment containing a human H3 genomic clone [6] (Fig. la). Densitometric scanning of autoradiograms showed an important (= 6-fold) and sustained increase in the COII mRNA levels after 48 h in culture coinciding with the entry of the bulk of thymocytes into the S-phase as determined by the [3H]thymidine uptake and by the expression of the histone H3 gene (Fig. lb). The fact that H3 mRNA was detected at relatively high levels in unstimulated cells (0 h) (Fig. la), is a consequence of thymocytes, unlike other cells, maintaining a large pool of proliferating imnlature blasts within the thymus [22,23]. Moreover, the decrease in the amount of H3 mRNA during the first 24 h is in accordance with the pattern of thymidine incorporation (Fig. lb).

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t ime (hou rs) Fig. 1. (a) RNA from rat thymocytes was collected at various times (0, 12, 24, 48 and 96 h) after stimulation with ConA plus rlL2. l0 Aagof total RNA from the stimulated thymocytes were subjected to Northern blot and hybridized with the COIl and H3 probes under the conditions described elsewhere [3]. Before rehybridization, the filters were stripped of radioactivity by boiling two times in 0.1~ SDS, 5 mM EDTA for 15 rain each. (b) 10/~g of total RNA from either rat thymocytes freshly obtained (0 h) or maintained 24 h in culture without stimulation, were analysed as indicated above. Ethidium bromide staining (lower panels) was used to check for RNA integrity and equal loading. Exposure times were 9 h for COII and two days for H3. (c) Changes in COIl (o) and H3 (o) mRNA levels and [3H]thymidine uptake (z~) during thymocyte proliferation and growth arrest (inset). Thymidine uptake was measured by pulsing parallel 200/zl cultures (4.105 cells) with 1/~Ci of [3HJthymidine: the time-point 0 h was determined by pulsing fresh thymocytes for 20 h without stimulation; the time-point 12 h was evaluated by pulsing stimulated thymocytes during only 12 h and the other time-points were determined by adding tritiated thymidine during the last 20 h of the culture period. The incorporation of radioactivity into DNA was determined by liquid scintillation counting. The mRNA levels are expressed as a percent of the maximal level obtained by scanning densitometry and plotted against time.

To further characterize the relationship between COII gene expression and cell proliferation, freshly isolated thymocytes were also cultured during 24 h without

mitogenic stimulation since these culture conditions promote a total decrease in their spontaneous proliferative activity [22]. To this end, total RNA was prepared

186 from these cells and hybridized with the C O ! I and H3 probes (Fig. lb). The densitometric analysis showed a 4.5-fold decrease in the C O I I m R N A levels coinciding with the decline in the proliferative activity, as indicated

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Fig. 2. (a) 10 ~g of total RNA, prepared at the indicated times (0, 12, 24, 48, 72 and 96 h) after ConA stimulation of rat splenic T-cells, was hybridized with the COIl and H3 probes. (b) 20/~g of total RNA, extracted after surgery from regenerating livers at the indicated times (0, 6, 12, 24, 28, 32, 36 and 48 h), were hybridized as indicated in above. Northern blot analyses were carried out as indicated in Fig. 1. Lower panels show ethidium bromide staining of 18S rRNA.

187 gesting that an increment in the oxidative metabolism is needed during the proliferation of these thymic cells. To verify whether this correlation between COII mRNA levels and cellular proliferation was extensive to other cell types, we examined the evolution of the COIl mRNA during the cell cycle of splenic T-cell populations as well as during hepatic regeneration after partial hepatectomy. Total rat splenic cells were cultured as indicated above and stimulated with 6 / t g / m l of ConA, a T-cell specific polyclonal activator [7]. For hepatectomy, 1-month-old rats were fasted overnight and between 9-11 a.m. subjected to 70~ hepatectomy [8]. At different times after operations, livers were removed, immediately frozen in liquid N2 and stored at - 7 0 ° C until RNA preparation. Total RNA was prepared from ConA-stimulated splenic T-cells and hybridized to the COIl and H3 probes. As shown in Fig. 2a, the COII mRNA levels remained nearly constant throughout the entire cell cycle while H3 mRNA increased sharply between 48-72 h, matching with the maximum of [3H]thymidine uptake (data not shown). Similar results were obtained for the COII mRNA during the rat liver regeneration (Fig. 2b). Under these experimental conditions, H3 mRNA was maximally expressed between 24-36 h indicating that hepatocytes reached the S-phase. These results are in good agreement with the constant COII mRNA levels reported during the proliferation of fibroblasts [5] and CTLL-2 cells [9]. One possible interpretation for our results concerning the variations in the levels of the COII message in three different proliferative systems, is that in general an increment in the oxidative metabolism during cell proliferation is not always necessary. The elevation of COII mRNA in proliferating thymocytes is striking. An explanation for this finding could be based on their unusually high levels of deoxynucleoside triphosphates (dNTPs) [10]. The dNTPs rise in the S-phase of thymocytes [10] and are essential for DNA synthesis as well as for the activity of the terminal deoxynucleotidyltransferase, an enzyme that appears only in immature B- and T-cells [11,12]. We have previously proposed that the expression of COII could be linked to the maintenance of thymocyte dNTPs pool [3]. According to this, it is tempting to speculate that a high oxidative metabolism during thymocyte proliferation could be important for supplying the energy requirements that allows the rise of dNTPs in the S-phase. Several contradictory data have been reported about whether glycolysis or oxidative metabolism is responsible for supplying the majority of the energetic demands for cell proliferation. Thus, an increment in the cytochrome c oxidase activity was not necessary during the proliferation of lymphoid cells [13], several cell lines [14-16], turnouts [14] and liver regeneration [17]. How-

ever, other authors reported opposite results during the same processes [18-21]. In this sense, our findings are in good agreement with the supposition that a rise in the glycolysis is enough to provide most of the energy for cellular proliferation. Clearly, the study of the variations in the activity of this and other cytochromes in the mitochondrial respiratory chain, both at the enzyme and gene levels, will help to clarify the ambiguity between energy requirement provided by glycolysis and oxidative metabolism. This work was supported by a grant from the Spanish Ministerio de Sanidad y Consumo (FISss 89/0481). The authors are gratefull to Drs. N. Glaichenhaus and J. Stein for providing the COII and H3 probes respectively. A.O. and X.R.B. are recipients of a F.P.I. fellowship from the Spanish government. References i Clayton, D.A. (1984) Annu. Rev. Biochem. 53, 573-5~4. 2 Chart, S.I. and Li, P.M. (1990) Biochemistry 29, 1-12. 3 Bustelo, X.R., Pichel, J.G., Dosil, M., Segade, F. and GbmezM~lrquez, J. (1990) Biochim. Biophys. Acta 977, 341-343. 4 Schumperli, D. (1986) Cell 45, 471-472. 5 Glaichenhaus, N., Leopold, P. and Cuzin, F. (1986) EMBO J. 5. 1261-1265. 6 Plumb, M., Stein, J. and Stein, G. (1983) Nucleic Acids Res. 11, 2391-2410. 7 Di Sabato, G., Hall, J.M. and Thompson, L.A. (1987) Met: ods Enzymol. 150, 3-17. 8 Waynforth, H.B. (1980) in Experimental and Surgical Technique in the Rat, pp. 137-139, Academic Press, London. 9 Dautry, F., Well, D., Yu, J. and Dautry-Varsat, A. (1988) J. Biol. Chem. 263, 17615-17620. 10 Cohen, A., Barankiewicz, J., Lederman, H.M. and Gelfand, E.W. (1983) J. Biol. Chem. 258, 12334-12340. 11 Chang, L.M.S. and Bollum, F.J. (1986) Crit. Rev. Biochem. 21, 27-52. 12 Barton, R., Goldsehneider, I. and Bollum, F.J. (1976) J. Immunol. 116, 462-468. 13 Harrison, E.H., Zbuzek, V., Labadie, J. and Bowers, W.E. (1981) Biochim. Biophys. Acta 676, 321-328. 14 Sun, A.S. and Cederbanm, A.I. (1990) Cancer Res. 40, 4677-4681. 15 Sun, A.S., Aggarwal, B.B. and Packer, L. (1975) Arch. Biochem. Biophys. 170, 1-11. 16 Morals, R. and Giguere, L. (1979) J. Cell. Physiol. 101, 77-88. 17 Verity, M.A., Travis, G. and Cheung, M. (1975) Exp. Mol. Pathol. 22, 73-84. 18 Van den Bogert, C., Melis, T.E. and Kroon, A.M. (1989) J. Leuk. Biol. 46, 128-133. 19 Hardt. N., De Kegel, D., Vanheule, L., Villani, G. and Spadari, S. (1980) Exp. Cell. Res. 127, 269-276. 20 Van den Bogert, C., Dontje, B.H.J., Holtrop, M., Melis, T.E., Romijn, J.C., Van Dongen, J.W. and Kroon, A.M. (1986) Cancer Res. 46, 3283-3289. 21 Van den Bogert, C., Van Kemebeek, G., De Leij, L. and Kroon, A.M. (1986) Cancer Len. 32, 41-51. 22 Bustelo, X.R., Otero, A., G6mez-M:~rquez,J. and Freire, M. (1991) J. Biol. Chem., in press. 23 Adkins, B., Mueller, C., Okada, C.Y., Reichert, R.A., Weissman, I.L. and Sprangrude, G.J. (1987) Annu. Rev. Immunol. 5, 325-365.