JOURNAL OF CELLULAR PHYSIOLOGY 152632-638 (1992,
Relationship Between Culture Conditions and the Dependency on Mitochondria1 Function of Mammalian Cell Proliferation C. VAN D E N BOCERT,* J.N. SPELBRINK,
AND
H.L. DEKKER
E C Slaler Institute for Biochemical Research, University of Amsterdam, Academic Medicdl Centre, I 1 0 5 AL Amsterdam, The Netherlands In cultured mammalian cells, the relationship was investigated between mitochondrial function dnd proliferation under various culture conditions. Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division. Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division. This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP-generating capacity of the cells was limiting. Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed. Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO, levels, completely prevented proliferation arrest. Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media. Pyruvate and C 0 2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media. Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATPgenerating capacib of the cells i s not limiting. GI 1i192 WiIcv-Li$s, Inc.
Mitochondria contain a number of copies of small circular DNA molecules (mtDNA) that are transcribed and translated separately from nuclear DNA. Marnmalian mtDNA exclusively carries the genetic information for 13 polypeptides, all of which are subunits of enzyme complexes involved in oxidative phosphorylation (Nelson, 1987). Therefore, impairment of mitochondrial gene expression results in disturbances of ATP generation and/or intermediary metabolism. It is likely that the severity of these secondary effects depends on several factors. First, the capacity for oxidative phosphorylation varies between different tissues and cells, at least in vivo (Van den Bogert e t al., 1992). Differences in the degree to which the concentration of enzymes involved in oxidative phosphorylation can be reduced before secondary effects are observed arc therefore conceivable (Van den Bogert e t al., 1983). Second, external factors may be of importance, e.g., the availability of substrates from which ATP can be derived by glycolysis, or the availability of several compounds that are able to replace precursor molecules that normally result from mitochondrial intermediary metabolism. In line with this, for cultured cells a number of culture conditions are known to influence the extent to which proliferation depends on mitochondrial function. It has been shown that cultured mammalian cells derive their energy from both glgcolysis and oxidative phosphorylation (Zielke et al., 1978; Stanisz et al., 1983). Under certain conditions, however, they can obtain the energy required for proliferation either from glycolysis only or from the oxidation of glutamine (Wice C 1992 WILEY-LISS,
INC.
et al., 1981; Zielke et al., 1984). In the latter case purines and pyrimidines have to be added to replace the ribonucleotide precursors that normally are formed during glycolysis 1Zielke et al., 1976). It has also been demonstrated that avian cells and Chinese hamster embryo cells can grow in culture without functional mitochondria, provided that uridine is present (Morais and Giguere, 1979; Morais et al., 1980, 1988; Morais and Guertin, 1982). This can be explained by the synthesis of uridine precursors requiring a functional respiratory chain (Gregoirc e t al., 1984). Moreover, it has been shown that the presence of pyruvate reduces the growth inhibitory effects of antimitochondrial drugs in tissue culture (Howell and Sager, 1979; Harris, 1980; Howell and Lee, 1989) and stimulates the growth of mitochondrial mutants (Howell and Lee, 1989). Most likely, pyruvate serves a s a n alternate source of precursors for the synthesis of various compounds under these conditions (Howell and Sager, 1979; Whitfield, 1985).It has recently been demonstrated that human cells, devoid of mtDNA, can also grow in culture provided that pyruvate and uridine are both present (King and Attardi, 1989). This observation has gained much attention since i t offers various possibilities to study mitochondrial diseases in cell lines from patients (King and Attardi, 1988).
Received December 10,1991; accepted April 1,1992
*To whom reprint requests/correspondence should be addressed.
MITOCHONDRIA AND PROLIFERATJON OF MAMMALlAN CELLS
It is obvious that insight into the factors that allow the growth of cultured mammalian cells with severe mitochondrial dysfunction is important, not only for diagnostic purposes, but also in fundamental research. Therefore, we have performed a detailed study on the relationship between mitochondrial function and proliferation in several cell lines under different culture conditions. To influence the degree of mitochondrial function, mitochondrial gene expression was inhibited by doxycycline. This antibiotic selectively blocks mitochondrial protein synthesis N a n den Bogert et al., 1986a, b, 1988,1991)and therefore reduces the concentration of mitochondrially coded subunits of enzymes involved in oxidative phosphorylation by 50% at every cell division. The results show that the dependency on mitochondrial function varied when different culture conditions were used. However, under the same culture conditions not all cell lines showed the same growth response to inhibition of mitochondrial function. There is certainly a pyruvate dependency for growth of cells without functional mitochondria, but this was only seen under culture conditions where the ATP-generating capacity of the cells was not limiting.
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concentration of 20 . 106/ml. The cell suspension was divided into the several aliquots needed for various analytical purposes and stored at -80°C.
Analytical e s s a y s To analyse the growth kinetics, cells were cultured in small (25 cm2) culture flasks. The total number of cells per culture was calculated by counting the cells after trypsinisation with a known volume of trypsine and subsequent dilution with normal medium. Molt-4 cells were counted by taking a small sample from the total suspension culture. Cell counting was performed microscopically with the aid of a counting chamber. Two different samples from the same cell culture were counted. Moreover, for every time-point 2 independent cultures were analysed. Growth curves were constructed by dividing the number of cells per culture by the number used to inoculate the first culture of a n experiment. The resulting value was corrected for the several dilutions originating from splitting the cultures during the course of a n experiment. A given cell line was always inoculated a t the same density and volume after splitting a culture. The cell density was kept such that the growth of untreated cells remained exponential. The activities of 2 mitochondrial enzymes, cytochrome MATERIALS AND METHODS c oxidase and citrate synthase, were used as measures Cell lines for the effect of different culture conditions on the celluTo study cultured mammalian cells with different lar content of these enzymes. Moreover, since cydegrees of differentiation, the following mouse cell tochrome c oxidase contains 3 subunits encoded by lines were used. P19 EC, a mouse embryo-carcinoma mtDNA, the enzyme activities could also be used to stem cell line, was used as a model for undifferentiated, study the effect of DC. The activity of cytochrome c pluripotent stem cells (McBurney and Rogers, 1982). oxidase was measured spectrophotometrically at 20°C P19 MES represents a stable mesodermal cell line de- in 30 mM phosphate buffer (pH 7.41, using 14 pM borived from P19 EC (Mummery et al., 2986); i t served a s vine heart cytochrome c as substrate (Borst et al., a model for early mesodermal cells. Since fibroblasts 1967). The activity was expressed as the first-order reare of mesodermal embryonic origin, a n established fi- action rate constant, h, per min. Citrate synthase activbroblast cell line (3T3) was studied also. The estab- ity was also measured spectrophotometrically at 20°C lished mouse cell lines (P19 EC, P19 MES, and 3T3) (Srere, 19691, the activity being expressed as the were cultured on gelatin-coated flasks. Moreover, two amount of product formed per min. The activity of the 2 different human cell lines were used. The established enzymes was assessed after preincubation of the samhuman leukemia cell line M o b 4 served as a n example ples with 1.5%laurylmaltoside. The protein content of of poorly differentiated cells. A primary line of diploid the samples was estimated using a modified Lowry fibroblasts was used as a model for more differentiated method (Peterson, 1977). To analyse the energy charge of the cells under differhuman cells. The fibroblast line was initiated from a skin biopsy of a healthy individual and used after ent culture conditions, the ATP and ADP concentra10-20 passages in culture. Molt-4 cells were grown as a tions in Molt-4 cells were determined. A known number suspension culture and human fibroblasts as monolay- of cells was collected by centrifugation, washed with ers. All cell lines were cultured at 37°C in a humidified PBS and extracted for 24 h with 60% methanol at atmosphere. The culture media were refreshed twice a -20°C (Donofrio et al., 1978). The nucleotides in this extract were analysed by FPLC, using a mono-& colweek. To study the effect of inhibition of mitochondrial pro- umn. The nucleotides were separated by elution with tein synthesis, doxycycline (DC) was added to the cul- two successive lineair salt gradients. The first ranged tures to a final concentration of 15 pg/ml. DC was pre- from 4 mM KH,PO, (pH 5.6), 0 mM KC1 to 4.5 mM pared a s a stock solution of 6 mgiml in 70% ethanol. KH,PO, (pH 5.61, 100 mM KC1, the second from 4.5 Control cultures received the same amount of ethanol. mM KH,PO, (pH 5.6), 100 mM KC1 to 9 mM KH,PO, Molt-4 cells were collected by centrifugation of the (pH 5.6),250 mM KC1. Standard mixtures, containing cultures. All other cultured cells were trypsinised and known amounts of the nucleotides of interest, were after inactivation of trypsine by addition of complete used for calibration. culture medium, they too were collected by centrifugaRESULTS tion. The cells were washed twice with phosphate-buffFigure 1 shows the effect of continuous inhibition of ered saline (PBS; 140 mM NaC1, 27 mM KC1, 6.5 mM Na,HPO,, 1.5 mM KH,PO,, 1 mM EDTA). The cells mitochondrial protein synthesis on the proliferation of were resuspended in PBS supplemented with 10%glyc- 3 different mause cell lines, cultured in standard RPMI erol, counted in a counting chamber, and adjusted t o a 1640 medium a t a C 0 2 tension of 5% in air. This me-
VAN DEN BOGERT ET AL
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6-
5-
4v)
c .-0 .L" 3 .-> rn
I
20
60
100
140
hours o f c u l t u r i n g
Fig. 1. Effect of inhibition of mitochondrial protein synthesis on the proliferation of P19 EC, P19 MES, and 3T3 cells. Cells were cultured in RPMI in the absence (closed symbols) or the presence (open symbols) of 15 kg DC per ml. The growth was followed by registering the cell number in a counting chamber after trypsinisation of the cells in individual culture flasks at the time-points indicated. The number of divisions was calculated and used as a measure for the growth rate. .:P19 EC; A :P19 MES; C3T3.
dium was supplemented with 10% fetal calf serum (FCS) and 2.2 g of NaHCO, per litre to obtain a pH of 7.4 at a CO, tension of 5%.RPMI 1640 contains 11mM glucose, 2 mM glutamine, and no pyruvate. Control cultures of the 3 cell lines showed large variations in doubling time, ranging from about 10 h for P19 EC to about 24 h for 3T3 cells. These differences most likely reflected the different degrees of differentiation of the respective cell lines: a low level of differentiation being accompanied by a fast proliferation rate. Inhibition of mitochondrial protein synthesis resulted in proliferation arrest after 1cell division in the case of P19 MES and 3T3 cells. About 5-6 cell divisions were possible for P19 EC cultures under these conditions. As shown before (Van den Bogert et al., 1992), significant differences in the specific activities of mitochondrial enzymes were not observed between the 3 cell lines. Moreover, DC treatment resulted in all 3 cell lines in a reduction by 50% of the specific cytochrome c oxidase activity a t every cell division (results not shown). Proliferation of P19 EC cells in RPMI was thus only affected when the concentration of subunits of mitochondrial genetic origin was reduced to about 3% of the normal value. In contrast, in the 2 other cell lines a content of mitochondrially synthesised proteins of about 50% was apparently already a critical limit for Proliferation. The effect of continuous inhibition of mitochondrial protein synthesis was also studied culturing the same 3 cell lines in Dulbecco's Modified Eagle (DME) medium,
containing 5.5 mM glucose, 4 mM glutamine, and 1mM pyruvate (referred to here a s DMEa). This medium was also supplemented with 10% FCS and 2.2 g of NaHC03 per litre and used at a CO, tension of 5%. Using this medium, proliferation arrest was not observed. After about 5 cell divisions, the growth rate became less, but the cells continued to proliferate a t a constant, reduced rate (results not shown). The content of mitochondrial enzymes in untreated cultures was, however, comparable to that of control cells cultured in RPMI, a s was the effectivity of DC treatment. Essentially the same results were found when a primary line of normal human fibroblasts was cultured in the presence of DC in RPMI or DMEa. In RPMI, proliferation was arrested after 1 cell doubling. DC treatment of cells cultured in DMEa led to a n extension of the doubling time after 2-3 generations, but did not result in proliferation arrest. However, culturing these cells a t a CO, tension of 5% in another standard type of DME medium, containing 25 mM glucose but no pyruvate (referred to here as DMEb), supplemented with 10% FCS and 2.2g of NaHCO, per litre, resulted in proliferation arrest after about 5 divisions. To analyse the dependency on mitochondrial function under different culture conditions in more detail, the proliferation of Molt-4 cells, leukemic cells of human origin, was studied. Figure 2 shows the effect of continuous inhibition of mitochondrial protein synthesis on the growth of the cells cultured in media based on RPMI or DME, a t either 5 or 10% C02. The amount of NaHCO, added was adjusted to 3.7 g per litre in the case of culturing at a CO, tension of 10%. The growth kinetics of the control cultures were not influenced by the type of medium used. However, the cells were only able to proliferate continuously in the presence of DC in DME media if both pyruvate was present and the CO, tension was raised to 10%. In fact, for these cells the CO, tension was more important than the presence of pyruvate, as can be deduced from the different growth curves of cells cultured in DME containing pyruvate, a t 5 and 10% CO,, respectively. Proliferation in the presence of both DC and pyruvate appeared only to be possible during 2 cell generations a t a CO, tension of 2% (results not shown). The glucose concentration was not of essential importance, though a higher concentration allowed the cells to proliferate faster during the initial cell doublings in the presence of DC. None of these conditions influenced the effect of inhibition of mitochondrial protein synthesis when M o b 4 cells were cultured in RPMI. Regardless of the CO, tension, the glucose or the pyruvate concentrations, the cells stopped to proliferate after 1 division. This implies that 1or 2 single factors are not decisive for the capability of mammalian cells to proliferate with a reduced content of functional mitochondria. Since there are no compounds present in standard DME that are absent in RPMI, a single factor can likewise not account for the different effect on proliferation during inhibition of mitochondrial protein synthesis of the same cell line cultured in either DME or RPMI. The difference between RPMI and DME, which might be of relevance in this context, is the higher concentration of almost all amino acids and vitamins in DME. It is conceivable that cells grown in DME can dispense with
MITOCHONDRIA AND PROLIFERATION OF MAMMALIAN CELLS
I
0
I
I
2
4
I
I
6 8 10 days of culturing
12
I
I
I
I
14
16
Fig. 2. Effect of inhibition of mitochondrial protein synthesis on the proliferation of Molt-4 cells cultured under various conditions. The cells were cultured in the absence (dotted line) or the presence of 15 pg DC per ml. The growth was followed by registering the cell number in a counting chamber after sampling a small volume from the suspension cultures. The number of divisions was calculated and used as a measure for the growth rate. Each growth curve was constructed from the curves obtained in several independent experiments. 0:standard RPMl(11mM glucose, no pyruvate, 5% CO,); A : RPMI, 25 mM glucose; V : RPMI, 25 mM glucose, 1 mM pyruvate; m: RPMI, 25 mM glucose, 1 mM pyruvate, 10% CO,. A : standard DMKb (25 mM glucose, no pyruvate, 5% CO ), V : DMEb, 1 mM pyruvate; m: DMEb, 1 mM pyruvate, 10% CO,. standard DMEa (5.5 mM glucose, 1 mM pyruvate, 5% CO,); @: DMEa, 10% CO,.
6I
mitochondria1 ATP generation, whereas cells cultured in RPMI cannot, since their need for ATP is less. To investigate this, we compared the steady-state ATP and ADP concentrations of cells cultured under various conditions. Table 1 shows that there were no significant differences in the ATP and ADP contents between control cells cultured in different media. The ATP content of cells cultured in RPMI did, however, rapidly decrease during inhibition of mitochondrial protein synthesis. Simultaneously, the ADP levels became elevated. This points to a n increasing shortage of ATP-generating capacity: ATP consumption and production are not in balance any more and ADP accumulates. These changes preceded proliferation arrest and are therefore the most likely cause of inhibition of growth. After longer peri-
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ods of treatment, both the ATP and ADP concentrations decreased and this was accompanied and followed by cell death. In cells grown in DME, no effect on the cellular ATP or ADP levels was found during the first 3 cell divisions in the presence of DC. Thus a t a much lower concentration of oxidative phosphorylation enzymes that are partly mitochondrially synthesised, cells cultured in DME did not show signs of ATP shortage, whereas this was clearly seen in cells with a higher residual content of the enzymes but were cultured in RPMI. After longer periods of treatment, the ATP level did also decrease in cells cultured in DME. In the absence of pyruvate or at a CO, tension of 5%, i t declined after about 7 days of inhibition of mitochondrial protein synthesis and cell death occurred. In the presence of pyruvate and at a CO, tension of lo%, the ATP level decreased to about 50% of the normal value after long-term treatment, and the ADP level increased about 2-fold. This resulted in an ATP to ADP ratio of about 2.5, instead of a ratio of 10 as found in control cells. Figure 3 shows the activity of 2 mitochondrial enzymes and the protein content of cells cultured in the presence of DC in different media. The cytochrome c oxidase activity decreased, a s expected, by 50% at every cell doubling. After proliferation arrest, the activity remained more or less constant until cell death became apparent. The activity of citrate synthase, a nuclearly coded enzyme, closely followed the ATP content; in dying cells the enzyme was apparently more rapidly degraded than cytochrome c oxidase. The protein content of the cells also showed changes: in cells treated long term that remained proliferating, it decreased. As the citrate synthase activity and the ATP concentration showed the same degree of reduction per cell, this implies that these parameters remained normal per mg of protein.
DISCUSSI 0N In general, it appears that mammalian cells, cultured in RPMI are no longer able to proliferate if their content of mitochondrially coded polypeptides, and therefore the activity of 4 of the 5 enzyme complexes involved in oxidative phosphorylation, is reduced to about 50%. This has been shown before for primary rat fibroblasts and a rat sarcoma cell line (Van den Bogert e t al., 1986131, for human Molt-4 cells (Van den Bogert et al., 1988), and in this study for primary human fibroblasts and 2 mouse cell lines. Also in other culture media, comparable to RPMI with regard to the concentration of amino acids and vitamins, this appears to be the case, e.g., for BHK cells in GME (Glasgow’s Modification of Eagle) medium (Leezenberg et al., 1979) and for several human renal and prostate cell lines cultured in ME (Minimal Essential) medium (Van den Bogert et al., 1986a). In relatively poor culture media, mitochondrial function is therefore indispensable for proliferation. The data on the effect of inhibition of mitochondrial protein synthesis on the ATP and ADP levels of the cells, presented in this study and in more detail in a previous one (Van den Bogert e t al., 19881, strongly point to a lack of ATP-generating capacity as the primary cause of proliferation arrest. From another study (B.H. Groen et al.,
VAN DEN BOGERT ET AL
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TABLE 1. ATP and ADP concentrations of Molt-4 rells cultured in the presence of DC under various conditions Mrdium tvne
Period of DC treatment (days!
0 [control) 0.1 0.2 0.5 1.0 1.5 2.0 3.0 4.0 6.0 7.0 9.0 16.0 23.0 31.0 38.0 46.0 53.0 60.0
DME
RPMI 1640 (70% CO,) ArP ADP 3.99 (341 4.00 (2) 4.12 (2) 2.24 ( 2 ) 2 11 (3) 1 4 0 (3) 0 86 (3) 0.02 ( 2 ) n.d. ~~
0.45 (28) 0.39 ( 2 ) 0.48 (2) 0.Y6 ( 2 ) 1 04 (31 0 68 (3) 0 13 (3) 0.01 ( 2 ) n.d.
DME
(5%C0,i
(10% CO,) ~
ATP
ADP
ATP
ADP
3.82 (21)
0.39 (17)
3.95 (351
0.37 (35)
3 77 (3)
0 36 (3)
3 85 (2)
0 38 ( 2 )
4 01 (I) 3.81 (3) 3.76 (2) 2.91 (2) 0.12 (2, n.d.
0 44 (1) 0.34 (3) 0.38 (2) 0.35 (2) 0.02 (2) n.d.
3 93 (1)
0 39 ( I )
3.68 (2) 2.94 (2) 2.84 (3) 2.30 12) 2.00 (1) 1.82 (1) 1.83 i l l 1.89 (1) 1.93 (1) 1.89 (1) 2.05 (11
0 37 ( 2 , 0 45 (2) 0 74 ( 3 ) 0 63 (21 0 58 (11 0 56 (11 0 78 (11 0 59 (11 0 62 (IJ 0 72 (11 0 81 (1)
The ADP and ATP concentrations are expressed i n ninol per lo6 cells. ‘The mean value of several independent samples is given. The number of samples is shown between brackets. In the control samples, the s.e. did not exceed 1% uf the mean value. The media contained I mM pyriivaie and 25 mM glucose.
in preparation) we know that, regardless of the culture conditions, mitochondrial ATP production is almost zero if Molt-4 cells have been cultured during 1-2 cell doublings in the presence of DC. After this period, further proliferation in the presence of DC will thus only be possible if the requirement for ATP is met by glycolysis alone. As judged by the amount of lactate formed, the rate of glycolysis is enhanced during inhibition of mitochondrial protein synthesis in cultures in RPMI as well as in those in DME. From our data it follows that an enhanced glycolysis can compensate for the loss of ATP-generating capacity if the cells are cultured in DME, but not if they are cultured in RPMI. An exception to this general rule are P19 EC cells. Cultured in RPMI, these cells were able to perform about 5 cell divisions in the presence of DC before proliferation was arrested. There are no differences in the content of mitochondrial enzymes between P19 EC cells and the other 2 mouse cell lines used in this study (Van den Bogert et al., 1992). Despite the same activity of mitochondrial enzymes, such as cytochrome c oxidase and citrate synthase in the control cells, the different mouse cell lines appeared therefore to have different dependencies on mitochondrial functions. Since the oxidative phosphorylation capacity could be reduced to 2-39 of the normal value before proliferation of P19 EC cells was arrested, this implies that these cells can proliferate almost without mitochondrial ATP supply, even in RPMI. This might reflect a general trait of very early embryonic cells, namely, a large independency of the cells on mitochondrial function (Piko and Chase, 1973; Piko and Taylor, 1987). P19 EC cells responded to inhibition of mitochondrial protein synthesis when cultured in RPMI a s the other cell lines did when cultured in DME. This leads to the conclusion that cultured cells can do without mitochondrial ATP production if glycolysis can provide the ATP
required, either because the cells are grown in “rich” media (such as DME), which apparently reduces the need for ATP, or because the cells concerned have a n inherently low need for ATP (as might be the case in P19 EC cells). Nonetheless, even where glycolysis alone can satisfy the need for ATP, all cell lines stopped dividing in the absence of pyruvate when mitochondrial protein synthesis was continuously inhibited. This was always observed after 5-6 divisions of cultures grown in the presence of DC. Under these conditions, functional respiratory chains will hardly be present any more. As has been shown before, this will impair the synthesis of uridine (Gregoire et al., 1984).It has, however, also been shown that undialysed FCS, as used in our study, contains sufficient uridine to prevent the effects of lack of uridine synthesis (Karle et al., 1984). In line with this, we found that addition of extra uridine to the cultures did not prevent proliferation arrest. Another consequence of the absence of functional respiratory chains will be the accumulation of NADH inside the mitochondria. It is highly unlikely that this can be completely re-oxidised via glycolysis (Robinson e t al., 1990). Accumulation of NADH will result in impairment of several metabolic pathways inside the mitochondria because of feedback inhibition. This might be the reason why pyruvate is able to prevent proliferation arrest under these conditions. It has been shown for many cultured cell lines that glutamine, apart from being a n important energy source, is also indispensable as a precursor of for instance aspartate, which in turn is a n important precursor in the synthesis of several amino acids and ribonucleotides (McKeehan, 1982; Moreadith and Lehninger, 1984; Brand et al., 1986; Lanks et al., 1988). The formation of aspartate from glutamine will, however, be severely impaired if mitochondrial NADH levels are high. At least theoretically, it is con-
MITOCHONDRIA AND PROLIFERATION OF MAMMALIAN CELLS
0
3
6
3 .6
9
days o culturing in the
Fig. 3. Activity of mitochondrial enzymes, protein, and ATP content of Molt-4 cells cultured in the presence of DC under various conditions. The respective values are given as percentages of the control values, and represent the mean ofthe percentages found in at least 3 different experiments. A : protein content 11008: 118 mg/lOgcells, n = 250); :. ATP content ( 1 00%: 4.0nmol/lO" cells, n = 90); 'I: cytochrome c oxidase activity (100%: 26\10' cells, n = 250); 0:citrate synthase activity (1008:4.8/10" cells, n = 50); 0:number of cell divisions. A. Cells cultured in RPMl(25 mM glucose, 1mM pyruvate, 10% (20,). B. Cells cultured in DME (25 mM glucose, 1mM pyruvate, 5%'CO,). C. Cells cultured in DME (26 mM glucose, 1mM pyruvate, 10% CO,).
ceivable that aspartate is formed from pyruvate via either oxaloacetate or malate under these conditions. The former route requires pyruvate carboxylase and aspartate-oxaloacetate transaminase as enzymes and the latter malic enzyme. For both pathways, CO, is also required. ,411 3 enzymes are known to be present in cultured mammalian cells (Donelly and Schleffler, 1976). This explanation is supported by extensive studies on Chinese hamster ovary cell mutants without a functional respiratory chain. Here, proliferation remained possible when pyruvate or aspartate were added to the culture medium, and moreover these cells showed CO, dependency (Whitfield, 1985). At least for Molt4 cells, we also found a strong dependency on the C 0 2tension for growth. It is very likely that this dependency also exists for the other cell lines, since CO, will hardly be produced intracellularly if mitochondrial intermediairy metabolic pathways are inhibited. A CO, tension of 5% was apparently sufficient for the other cell lines; in M o b 4 cell cultures it had to be raised to 10% to allow proliferation to continue. This might be
637
related to the fact t h a t Molt-4 cells grow in suspension: the number of cells per culture in this case is substantially higher than in cultures of monolayers. In line with this assumption, Molt4 cells cultured for a prolonged time in the presence of DC could only be kept in a proliferating state by keeping the cells at low densities. Taken together, the results show that cultured mammalian cells are able t o survive without functional mitochondria if their ATP demand is reduced such that it can be supplied by glycolysis only and if pyruvate is present a s a n alternate precursor for compounds that are normally produced via mitochondrial metabolism. If these conditions have been fulfilled, the cells are apparently able to adjust their growth rate, size, and energy charge and remain proliferating. These conditions are highly unlikely to be met under more physiological conditions: in our previous work using several tumors as systems in vivo, inhibition of mitochondrial protein synthesis always resulted in proliferation arrest after 1-2 cell divisions (Van den Bogert et al., 1981, 1986a,b). Nonetheless, we feel the present data improve our insight in the relationship between mitochondria] function and proliferation of cultured cells. The data offer a number of explanations for apparently contradictory conclusions in the literature, e.g., on the question of whether or not cultured cells depend on mitochondrial ATP synthesis. In the majority of the cases, the answer will depend on the type of medium used. More important, our data are of value for the selection and maintenance of lines of cells t h a t are deficient with respect to 1 or more enzymes involved in oxidative phosphorylation, e.g., cell lines derived from patients with mitochondrial myopathies.
ACKNOWLEDGMENTS The members of theme 3 of the E.C. Slater Institute for Biochemical Research, University of Amsterdam are thanked for their cooperation and hospitality during many of the cell culture experiments. We also thank Drs. P.A. Bolhuis, N.H. Herzberg (Department of Neurology, University of Amsterdam1 and A.J. Meijer (E.C. Slater Institute for Biochemical Research) for their interest. Part of this study was made possible by a fellowship of C.V.d.B. of the Royal Netherlands Academy of Arts and Sciences, and a grant from "Het Prinses Beatrix Fonds," The Hague, The Netherlands.
LITERATURE CITED Borst, P., Ruttenberg, G.J.C.M., and Kroon, A.M. (1967) Mitochondrial DNA I. Preparation and properties of mitochondrial DNA from chick liver. Biochim. Biophys. Acta, 149t140-155. Brand, K., Leibold, W., Luppa, P., Schoerner, C . ,and Schulz, A. (1986) Metabolic alterations associated with proliferation of rnitogen-activated lymphocytes and of lymphoblastoid cell lines: Evaluation of glucose and glutamine metabolism. Immunobiol., 173:23-34. Donelly, M., and Schleff'ler, I.E. 11976)Energy metabolism in respiration-deficient and wild tvue Chinese hamster fibroblasts in culture. J. Cell. Physiol., 89:39-k%. Ilonofrio, J., Coleman, M.S., Hutton, cJ.J.,Daoud, A., Lampkin, B., and Dyminski, J . (1978) Overproduction of adenine deoxynucleosides and deoxynucleotides in adenosine deaminase deficiency with severe combined immunodeficiency disease. J. Clin. Invest., 62:884887. Gr(.goire, M., Morais, R., Quillam, M A . , and Gravel, D. (1984) On auxotrophy for pyrimidines of respiration-deficient chick embryo cells. Eur. J . Biochem., 142t49-55.
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Harris, M. (1980) Pyruvatc blocks cxpression of sensitivity to antimycin A and chloramphenicol. Som. Cell Genet., 6:699-708. Howell, N., and Lee, A. (1989) Sequence analysis of mouse mitochondrial chloramphenicol-resistant mutants. Som. Cell Genet., 25t237244. Howell, N., and Sager, R. (1979) Cytoplasmic genetics of mammalian cells: conditional sensitivity to mitochondrial inhibitors and isolation of new mutant phenotypes. Som. Cell Genet., FitS33-845. Karle, J.M., Anderson, L.W., and Cysyk, R.L. (1984) Effcct of plasma concentrations of uridine on pyrimidine biosynthesis in cultured L1210 cells. J . Biol. Chem., 259:67-72. King, M.P., and Attardi, G. (1988) Injection of mitochondria into human cells leads to a rapid replacement of the endogeneous mitochondrial DNA. Cell, 52t811-819. King, M.P., and Attardi, G. (1989)Human cells lacking mitochondrial DNA: 'depopulation with exogenous mitochondria by complementation. Science,246:500-503. Lanks, K.W.; and Li, P-W. !1988) End products of glucose and glutamine metabolism by cultured cell lines. J. Cell. Physiol., 235t151~ 155. Leezenberg, J.A., Wesseling, H., and Kroon, A.M. (1979)Possible cytostatic action of tetracyclines in the treatment of tumors of the nasopharynx and larynx. Eur. J . Clin. Pharmacol., 26237-241. McBurney, M., and Rogers, B.J. (1982) Isolation of male embryonal carcinoma cells and their chromosome replication patterns. Dev. Biol., 89503-508. McKeehan, W.L. (1982) Glycolysis, glutaminolysis and cell proliferation. Cell Biol. Tnt. Rep.. 6.535-651. Morais, R., and Giguere, L. (1979) On the adaptation of cultured chick embryo cells to growth in the presence of chloramphenicol. J. Cell. Physiol., 101:77-88. Morais, R., Gregoire, M., Jeanottc, L., and Gravel, D. 11980) Chick embryo cells rendered respiration deficient by chloramphenicol and ethidium bromide are auxotrophic for pyrimidines. Biochem. Biophys. Res. Comm., 94t71-77. Morais, R., Guertin, D., and Kornblatt, J.A. (1982) On the contribution of the mitochondrial genome to the growth of Chinese hamster embryo cells in culture. Can. J. Biochem., 60t290-294. Morais, R., Desjardins, P., Turmel, C., and Zinkewich-Pkotti, K. (19881 Development and characterization of continuous avian cell lines depleted of mitochondrial DNA. In Vitro Cell. Dev. Biol., 24,549658. Moreadith, R.W., and Lehninger, A.L. (1984) The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. J. Biol. Chem., 259t62154221. Mummery, C.L., Feyen, A,, Van den Brink, S., Molenaar, W.H., and De Laat, S.W. (1986) Establishment of a differentiated mesodermal line from P19EC cells expressing functional PDGF and EGF-receptors. Exp. Cell Res., 165329-242. Nelson, B.D. (1987) Biogenesis of mammalian mitochondria. In: Current Topics in Bioenergetics, Vol. 15. C.P. Lee; ed. Academic Press, San Diego, pp. 221-272. Peterson, G.L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem., 831346-356. Piko, L., and Chase, D.G. (1973) Role of the mitochondrial genome during early development in mice. Effects of ethidium bromide and chloramphenicol. J. Cell Biol., 58,357378.
Pik6, L., and Taylor, K.D. (1987)Amounts of mitochondrial DNA and abundance of some mitochondrial gene transcripts i n early mouse embryos. Dcv. Biol., 123336L374. Robinson, B.H., Glerum, D.M., Chow, W., Petrova-Benedict, R., Lightowlers, R., and Capaldi, R. (1990) The use of skin fibroblasts cultures in the detection of respiratory chain defects in patients with lacticacidemia. Pediatr. Res., 28t549-555. Srere, P.A. (1969)Citrate synthase. In: Methods in Enzymology, Vol. 13. J.M. Lowenstein, ed. Academic Press, London, pp. 3-26. Stanisz, J., Wice, B.M., and Kennell, D.E. 11983)Comparative energy metabolism in cultured heart muscle and Hela cells. J . Cell. Physiol., 115t320-330. Van den Bogert, C., Dontje, B.H.J., Wijbenga, J.J., and Kroon, A.M. (1981) Arrest of in vivo proliferation of Zajdela tumor cells by inhibition of mitochondrial protein synthesis. Cancer Res., 41.19431947. Van den Bogert, C., Lont, M., Mojet, M., and Kroon, A.M. (1983) Impairment of liver regeneration during inhibition of mitochondrial protein synthesis by oxytetracycline. Biochim. Biophys. Acta, 722r393-400. Van den Bogert, C., Dontje, B.H.J., Holtrop, M., Melis, T.E., Romijn, J.C. Van Dongen, J.W., and Kroon, A.M. (1986a) Arrest of the proliferation of renal and prostate carcinomas of human origin by inhibition of mitochondrial protein synthesis. Cancer Res., 46t32833289. Van den Bogert, C., Van Kernebeek, G., De Leij, L., and Kroon, A.M. (1986b)Inhibition of mitochondrial protein synthesis leads to proliferation arrest in the G1-phase ofthe cell cycle. Cancer Lett., 32r4151. Van den Bogert, C., Muus, P., Haanen, C., Pennings, A,, Melis, T.E., and Kroon, A.M. (1988)Mitochondrial biogenesis and mitochondrial activity during the progression of the cell cycle of human leukemic cells. Exp. Cell Res., 178:143-153. Van den Bogert, C., Pennings, A,, Dekker, H.L., Boezeman, J.H.M., Luciakova, K., and Sinjorgo, K.C.M. (1991) Quantification of mitochondria] proteins in cultured cells by immuno-flow cytometry. Biochim. Biophys. Acta, 1097r87-94. Van den Bogert, C., Dekker, H.L., Cornelissen, J.C., Van Kuilenburg, A.B.P., Bolhuis, P.A., and Muijsers, A.O. (1992) Isoforms of cytochrome c oxidase in tissues and cell lines of the mouse. Biochim. Biophys. Acta, 1099:11%122. Whitfield, C.D. (1985)Mitochondrial mutants. In: Molecular Cell Genetics. M.M. Gottesmann, ed. John Wiley & Sons, New York, pp. 545-689. Wicc, B.M., Reitzer, L.J., and Kennell, D. 119811 The continuous growth of vertebrate cells in the absence of sugars. J. Biol. Chem., 265t7812-7819. Zielke, H.R., Ozand, P.T., Tildon, J.T., Sevdalian, D.A., and Cornblath, M. (1976) Growth of human diploid fibroblasts in the absence of glucose utilization. Proc. Natl. Acad. Sci. USA, 73t4110-4114. Zielke, H.K., Ozand, P.T., Tildon, J.T., Sevdalian, D.A., and Cornblath, M. (1978) Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts. J . Cell. Physiol., 95.41-48. Zielke, H.R., Zielke, C.L., and Ozand, P.T. (1984) Glutamine: A major energy source for cultured mammalian cells. Federation Proc., 43t121-125.
Relationship Between Culture Conditions and the Dependency on Mitochondria1 Function of Mammalian Cell Proliferation C. VAN D E N BOCERT,* J.N. SPELBRINK,
AND
H.L. DEKKER
E C Slaler Institute for Biochemical Research, University of Amsterdam, Academic Medicdl Centre, I 1 0 5 AL Amsterdam, The Netherlands In cultured mammalian cells, the relationship was investigated between mitochondrial function dnd proliferation under various culture conditions. Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division. Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division. This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP-generating capacity of the cells was limiting. Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed. Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO, levels, completely prevented proliferation arrest. Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media. Pyruvate and C 0 2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media. Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATPgenerating capacib of the cells i s not limiting. GI 1i192 WiIcv-Li$s, Inc.
Mitochondria contain a number of copies of small circular DNA molecules (mtDNA) that are transcribed and translated separately from nuclear DNA. Marnmalian mtDNA exclusively carries the genetic information for 13 polypeptides, all of which are subunits of enzyme complexes involved in oxidative phosphorylation (Nelson, 1987). Therefore, impairment of mitochondrial gene expression results in disturbances of ATP generation and/or intermediary metabolism. It is likely that the severity of these secondary effects depends on several factors. First, the capacity for oxidative phosphorylation varies between different tissues and cells, at least in vivo (Van den Bogert e t al., 1992). Differences in the degree to which the concentration of enzymes involved in oxidative phosphorylation can be reduced before secondary effects are observed arc therefore conceivable (Van den Bogert e t al., 1983). Second, external factors may be of importance, e.g., the availability of substrates from which ATP can be derived by glycolysis, or the availability of several compounds that are able to replace precursor molecules that normally result from mitochondrial intermediary metabolism. In line with this, for cultured cells a number of culture conditions are known to influence the extent to which proliferation depends on mitochondrial function. It has been shown that cultured mammalian cells derive their energy from both glgcolysis and oxidative phosphorylation (Zielke et al., 1978; Stanisz et al., 1983). Under certain conditions, however, they can obtain the energy required for proliferation either from glycolysis only or from the oxidation of glutamine (Wice C 1992 WILEY-LISS,
INC.
et al., 1981; Zielke et al., 1984). In the latter case purines and pyrimidines have to be added to replace the ribonucleotide precursors that normally are formed during glycolysis 1Zielke et al., 1976). It has also been demonstrated that avian cells and Chinese hamster embryo cells can grow in culture without functional mitochondria, provided that uridine is present (Morais and Giguere, 1979; Morais et al., 1980, 1988; Morais and Guertin, 1982). This can be explained by the synthesis of uridine precursors requiring a functional respiratory chain (Gregoirc e t al., 1984). Moreover, it has been shown that the presence of pyruvate reduces the growth inhibitory effects of antimitochondrial drugs in tissue culture (Howell and Sager, 1979; Harris, 1980; Howell and Lee, 1989) and stimulates the growth of mitochondrial mutants (Howell and Lee, 1989). Most likely, pyruvate serves a s a n alternate source of precursors for the synthesis of various compounds under these conditions (Howell and Sager, 1979; Whitfield, 1985).It has recently been demonstrated that human cells, devoid of mtDNA, can also grow in culture provided that pyruvate and uridine are both present (King and Attardi, 1989). This observation has gained much attention since i t offers various possibilities to study mitochondrial diseases in cell lines from patients (King and Attardi, 1988).
Received December 10,1991; accepted April 1,1992
*To whom reprint requests/correspondence should be addressed.
MITOCHONDRIA AND PROLIFERATJON OF MAMMALlAN CELLS
It is obvious that insight into the factors that allow the growth of cultured mammalian cells with severe mitochondrial dysfunction is important, not only for diagnostic purposes, but also in fundamental research. Therefore, we have performed a detailed study on the relationship between mitochondrial function and proliferation in several cell lines under different culture conditions. To influence the degree of mitochondrial function, mitochondrial gene expression was inhibited by doxycycline. This antibiotic selectively blocks mitochondrial protein synthesis N a n den Bogert et al., 1986a, b, 1988,1991)and therefore reduces the concentration of mitochondrially coded subunits of enzymes involved in oxidative phosphorylation by 50% at every cell division. The results show that the dependency on mitochondrial function varied when different culture conditions were used. However, under the same culture conditions not all cell lines showed the same growth response to inhibition of mitochondrial function. There is certainly a pyruvate dependency for growth of cells without functional mitochondria, but this was only seen under culture conditions where the ATP-generating capacity of the cells was not limiting.
633
concentration of 20 . 106/ml. The cell suspension was divided into the several aliquots needed for various analytical purposes and stored at -80°C.
Analytical e s s a y s To analyse the growth kinetics, cells were cultured in small (25 cm2) culture flasks. The total number of cells per culture was calculated by counting the cells after trypsinisation with a known volume of trypsine and subsequent dilution with normal medium. Molt-4 cells were counted by taking a small sample from the total suspension culture. Cell counting was performed microscopically with the aid of a counting chamber. Two different samples from the same cell culture were counted. Moreover, for every time-point 2 independent cultures were analysed. Growth curves were constructed by dividing the number of cells per culture by the number used to inoculate the first culture of a n experiment. The resulting value was corrected for the several dilutions originating from splitting the cultures during the course of a n experiment. A given cell line was always inoculated a t the same density and volume after splitting a culture. The cell density was kept such that the growth of untreated cells remained exponential. The activities of 2 mitochondrial enzymes, cytochrome MATERIALS AND METHODS c oxidase and citrate synthase, were used as measures Cell lines for the effect of different culture conditions on the celluTo study cultured mammalian cells with different lar content of these enzymes. Moreover, since cydegrees of differentiation, the following mouse cell tochrome c oxidase contains 3 subunits encoded by lines were used. P19 EC, a mouse embryo-carcinoma mtDNA, the enzyme activities could also be used to stem cell line, was used as a model for undifferentiated, study the effect of DC. The activity of cytochrome c pluripotent stem cells (McBurney and Rogers, 1982). oxidase was measured spectrophotometrically at 20°C P19 MES represents a stable mesodermal cell line de- in 30 mM phosphate buffer (pH 7.41, using 14 pM borived from P19 EC (Mummery et al., 2986); i t served a s vine heart cytochrome c as substrate (Borst et al., a model for early mesodermal cells. Since fibroblasts 1967). The activity was expressed as the first-order reare of mesodermal embryonic origin, a n established fi- action rate constant, h, per min. Citrate synthase activbroblast cell line (3T3) was studied also. The estab- ity was also measured spectrophotometrically at 20°C lished mouse cell lines (P19 EC, P19 MES, and 3T3) (Srere, 19691, the activity being expressed as the were cultured on gelatin-coated flasks. Moreover, two amount of product formed per min. The activity of the 2 different human cell lines were used. The established enzymes was assessed after preincubation of the samhuman leukemia cell line M o b 4 served as a n example ples with 1.5%laurylmaltoside. The protein content of of poorly differentiated cells. A primary line of diploid the samples was estimated using a modified Lowry fibroblasts was used as a model for more differentiated method (Peterson, 1977). To analyse the energy charge of the cells under differhuman cells. The fibroblast line was initiated from a skin biopsy of a healthy individual and used after ent culture conditions, the ATP and ADP concentra10-20 passages in culture. Molt-4 cells were grown as a tions in Molt-4 cells were determined. A known number suspension culture and human fibroblasts as monolay- of cells was collected by centrifugation, washed with ers. All cell lines were cultured at 37°C in a humidified PBS and extracted for 24 h with 60% methanol at atmosphere. The culture media were refreshed twice a -20°C (Donofrio et al., 1978). The nucleotides in this extract were analysed by FPLC, using a mono-& colweek. To study the effect of inhibition of mitochondrial pro- umn. The nucleotides were separated by elution with tein synthesis, doxycycline (DC) was added to the cul- two successive lineair salt gradients. The first ranged tures to a final concentration of 15 pg/ml. DC was pre- from 4 mM KH,PO, (pH 5.6), 0 mM KC1 to 4.5 mM pared a s a stock solution of 6 mgiml in 70% ethanol. KH,PO, (pH 5.61, 100 mM KC1, the second from 4.5 Control cultures received the same amount of ethanol. mM KH,PO, (pH 5.6), 100 mM KC1 to 9 mM KH,PO, Molt-4 cells were collected by centrifugation of the (pH 5.6),250 mM KC1. Standard mixtures, containing cultures. All other cultured cells were trypsinised and known amounts of the nucleotides of interest, were after inactivation of trypsine by addition of complete used for calibration. culture medium, they too were collected by centrifugaRESULTS tion. The cells were washed twice with phosphate-buffFigure 1 shows the effect of continuous inhibition of ered saline (PBS; 140 mM NaC1, 27 mM KC1, 6.5 mM Na,HPO,, 1.5 mM KH,PO,, 1 mM EDTA). The cells mitochondrial protein synthesis on the proliferation of were resuspended in PBS supplemented with 10%glyc- 3 different mause cell lines, cultured in standard RPMI erol, counted in a counting chamber, and adjusted t o a 1640 medium a t a C 0 2 tension of 5% in air. This me-
VAN DEN BOGERT ET AL
634
6-
5-
4v)
c .-0 .L" 3 .-> rn
I
20
60
100
140
hours o f c u l t u r i n g
Fig. 1. Effect of inhibition of mitochondrial protein synthesis on the proliferation of P19 EC, P19 MES, and 3T3 cells. Cells were cultured in RPMI in the absence (closed symbols) or the presence (open symbols) of 15 kg DC per ml. The growth was followed by registering the cell number in a counting chamber after trypsinisation of the cells in individual culture flasks at the time-points indicated. The number of divisions was calculated and used as a measure for the growth rate. .:P19 EC; A :P19 MES; C3T3.
dium was supplemented with 10% fetal calf serum (FCS) and 2.2 g of NaHCO, per litre to obtain a pH of 7.4 at a CO, tension of 5%.RPMI 1640 contains 11mM glucose, 2 mM glutamine, and no pyruvate. Control cultures of the 3 cell lines showed large variations in doubling time, ranging from about 10 h for P19 EC to about 24 h for 3T3 cells. These differences most likely reflected the different degrees of differentiation of the respective cell lines: a low level of differentiation being accompanied by a fast proliferation rate. Inhibition of mitochondrial protein synthesis resulted in proliferation arrest after 1cell division in the case of P19 MES and 3T3 cells. About 5-6 cell divisions were possible for P19 EC cultures under these conditions. As shown before (Van den Bogert et al., 1992), significant differences in the specific activities of mitochondrial enzymes were not observed between the 3 cell lines. Moreover, DC treatment resulted in all 3 cell lines in a reduction by 50% of the specific cytochrome c oxidase activity a t every cell division (results not shown). Proliferation of P19 EC cells in RPMI was thus only affected when the concentration of subunits of mitochondrial genetic origin was reduced to about 3% of the normal value. In contrast, in the 2 other cell lines a content of mitochondrially synthesised proteins of about 50% was apparently already a critical limit for Proliferation. The effect of continuous inhibition of mitochondrial protein synthesis was also studied culturing the same 3 cell lines in Dulbecco's Modified Eagle (DME) medium,
containing 5.5 mM glucose, 4 mM glutamine, and 1mM pyruvate (referred to here a s DMEa). This medium was also supplemented with 10% FCS and 2.2 g of NaHC03 per litre and used at a CO, tension of 5%. Using this medium, proliferation arrest was not observed. After about 5 cell divisions, the growth rate became less, but the cells continued to proliferate a t a constant, reduced rate (results not shown). The content of mitochondrial enzymes in untreated cultures was, however, comparable to that of control cells cultured in RPMI, a s was the effectivity of DC treatment. Essentially the same results were found when a primary line of normal human fibroblasts was cultured in the presence of DC in RPMI or DMEa. In RPMI, proliferation was arrested after 1 cell doubling. DC treatment of cells cultured in DMEa led to a n extension of the doubling time after 2-3 generations, but did not result in proliferation arrest. However, culturing these cells a t a CO, tension of 5% in another standard type of DME medium, containing 25 mM glucose but no pyruvate (referred to here as DMEb), supplemented with 10% FCS and 2.2g of NaHCO, per litre, resulted in proliferation arrest after about 5 divisions. To analyse the dependency on mitochondrial function under different culture conditions in more detail, the proliferation of Molt-4 cells, leukemic cells of human origin, was studied. Figure 2 shows the effect of continuous inhibition of mitochondrial protein synthesis on the growth of the cells cultured in media based on RPMI or DME, a t either 5 or 10% C02. The amount of NaHCO, added was adjusted to 3.7 g per litre in the case of culturing at a CO, tension of 10%. The growth kinetics of the control cultures were not influenced by the type of medium used. However, the cells were only able to proliferate continuously in the presence of DC in DME media if both pyruvate was present and the CO, tension was raised to 10%. In fact, for these cells the CO, tension was more important than the presence of pyruvate, as can be deduced from the different growth curves of cells cultured in DME containing pyruvate, a t 5 and 10% CO,, respectively. Proliferation in the presence of both DC and pyruvate appeared only to be possible during 2 cell generations a t a CO, tension of 2% (results not shown). The glucose concentration was not of essential importance, though a higher concentration allowed the cells to proliferate faster during the initial cell doublings in the presence of DC. None of these conditions influenced the effect of inhibition of mitochondrial protein synthesis when M o b 4 cells were cultured in RPMI. Regardless of the CO, tension, the glucose or the pyruvate concentrations, the cells stopped to proliferate after 1 division. This implies that 1or 2 single factors are not decisive for the capability of mammalian cells to proliferate with a reduced content of functional mitochondria. Since there are no compounds present in standard DME that are absent in RPMI, a single factor can likewise not account for the different effect on proliferation during inhibition of mitochondrial protein synthesis of the same cell line cultured in either DME or RPMI. The difference between RPMI and DME, which might be of relevance in this context, is the higher concentration of almost all amino acids and vitamins in DME. It is conceivable that cells grown in DME can dispense with
MITOCHONDRIA AND PROLIFERATION OF MAMMALIAN CELLS
I
0
I
I
2
4
I
I
6 8 10 days of culturing
12
I
I
I
I
14
16
Fig. 2. Effect of inhibition of mitochondrial protein synthesis on the proliferation of Molt-4 cells cultured under various conditions. The cells were cultured in the absence (dotted line) or the presence of 15 pg DC per ml. The growth was followed by registering the cell number in a counting chamber after sampling a small volume from the suspension cultures. The number of divisions was calculated and used as a measure for the growth rate. Each growth curve was constructed from the curves obtained in several independent experiments. 0:standard RPMl(11mM glucose, no pyruvate, 5% CO,); A : RPMI, 25 mM glucose; V : RPMI, 25 mM glucose, 1 mM pyruvate; m: RPMI, 25 mM glucose, 1 mM pyruvate, 10% CO,. A : standard DMKb (25 mM glucose, no pyruvate, 5% CO ), V : DMEb, 1 mM pyruvate; m: DMEb, 1 mM pyruvate, 10% CO,. standard DMEa (5.5 mM glucose, 1 mM pyruvate, 5% CO,); @: DMEa, 10% CO,.
6I
mitochondria1 ATP generation, whereas cells cultured in RPMI cannot, since their need for ATP is less. To investigate this, we compared the steady-state ATP and ADP concentrations of cells cultured under various conditions. Table 1 shows that there were no significant differences in the ATP and ADP contents between control cells cultured in different media. The ATP content of cells cultured in RPMI did, however, rapidly decrease during inhibition of mitochondrial protein synthesis. Simultaneously, the ADP levels became elevated. This points to a n increasing shortage of ATP-generating capacity: ATP consumption and production are not in balance any more and ADP accumulates. These changes preceded proliferation arrest and are therefore the most likely cause of inhibition of growth. After longer peri-
635
ods of treatment, both the ATP and ADP concentrations decreased and this was accompanied and followed by cell death. In cells grown in DME, no effect on the cellular ATP or ADP levels was found during the first 3 cell divisions in the presence of DC. Thus a t a much lower concentration of oxidative phosphorylation enzymes that are partly mitochondrially synthesised, cells cultured in DME did not show signs of ATP shortage, whereas this was clearly seen in cells with a higher residual content of the enzymes but were cultured in RPMI. After longer periods of treatment, the ATP level did also decrease in cells cultured in DME. In the absence of pyruvate or at a CO, tension of 5%, i t declined after about 7 days of inhibition of mitochondrial protein synthesis and cell death occurred. In the presence of pyruvate and at a CO, tension of lo%, the ATP level decreased to about 50% of the normal value after long-term treatment, and the ADP level increased about 2-fold. This resulted in an ATP to ADP ratio of about 2.5, instead of a ratio of 10 as found in control cells. Figure 3 shows the activity of 2 mitochondrial enzymes and the protein content of cells cultured in the presence of DC in different media. The cytochrome c oxidase activity decreased, a s expected, by 50% at every cell doubling. After proliferation arrest, the activity remained more or less constant until cell death became apparent. The activity of citrate synthase, a nuclearly coded enzyme, closely followed the ATP content; in dying cells the enzyme was apparently more rapidly degraded than cytochrome c oxidase. The protein content of the cells also showed changes: in cells treated long term that remained proliferating, it decreased. As the citrate synthase activity and the ATP concentration showed the same degree of reduction per cell, this implies that these parameters remained normal per mg of protein.
DISCUSSI 0N In general, it appears that mammalian cells, cultured in RPMI are no longer able to proliferate if their content of mitochondrially coded polypeptides, and therefore the activity of 4 of the 5 enzyme complexes involved in oxidative phosphorylation, is reduced to about 50%. This has been shown before for primary rat fibroblasts and a rat sarcoma cell line (Van den Bogert e t al., 1986131, for human Molt-4 cells (Van den Bogert et al., 1988), and in this study for primary human fibroblasts and 2 mouse cell lines. Also in other culture media, comparable to RPMI with regard to the concentration of amino acids and vitamins, this appears to be the case, e.g., for BHK cells in GME (Glasgow’s Modification of Eagle) medium (Leezenberg et al., 1979) and for several human renal and prostate cell lines cultured in ME (Minimal Essential) medium (Van den Bogert et al., 1986a). In relatively poor culture media, mitochondrial function is therefore indispensable for proliferation. The data on the effect of inhibition of mitochondrial protein synthesis on the ATP and ADP levels of the cells, presented in this study and in more detail in a previous one (Van den Bogert e t al., 19881, strongly point to a lack of ATP-generating capacity as the primary cause of proliferation arrest. From another study (B.H. Groen et al.,
VAN DEN BOGERT ET AL
636
TABLE 1. ATP and ADP concentrations of Molt-4 rells cultured in the presence of DC under various conditions Mrdium tvne
Period of DC treatment (days!
0 [control) 0.1 0.2 0.5 1.0 1.5 2.0 3.0 4.0 6.0 7.0 9.0 16.0 23.0 31.0 38.0 46.0 53.0 60.0
DME
RPMI 1640 (70% CO,) ArP ADP 3.99 (341 4.00 (2) 4.12 (2) 2.24 ( 2 ) 2 11 (3) 1 4 0 (3) 0 86 (3) 0.02 ( 2 ) n.d. ~~
0.45 (28) 0.39 ( 2 ) 0.48 (2) 0.Y6 ( 2 ) 1 04 (31 0 68 (3) 0 13 (3) 0.01 ( 2 ) n.d.
DME
(5%C0,i
(10% CO,) ~
ATP
ADP
ATP
ADP
3.82 (21)
0.39 (17)
3.95 (351
0.37 (35)
3 77 (3)
0 36 (3)
3 85 (2)
0 38 ( 2 )
4 01 (I) 3.81 (3) 3.76 (2) 2.91 (2) 0.12 (2, n.d.
0 44 (1) 0.34 (3) 0.38 (2) 0.35 (2) 0.02 (2) n.d.
3 93 (1)
0 39 ( I )
3.68 (2) 2.94 (2) 2.84 (3) 2.30 12) 2.00 (1) 1.82 (1) 1.83 i l l 1.89 (1) 1.93 (1) 1.89 (1) 2.05 (11
0 37 ( 2 , 0 45 (2) 0 74 ( 3 ) 0 63 (21 0 58 (11 0 56 (11 0 78 (11 0 59 (11 0 62 (IJ 0 72 (11 0 81 (1)
The ADP and ATP concentrations are expressed i n ninol per lo6 cells. ‘The mean value of several independent samples is given. The number of samples is shown between brackets. In the control samples, the s.e. did not exceed 1% uf the mean value. The media contained I mM pyriivaie and 25 mM glucose.
in preparation) we know that, regardless of the culture conditions, mitochondrial ATP production is almost zero if Molt-4 cells have been cultured during 1-2 cell doublings in the presence of DC. After this period, further proliferation in the presence of DC will thus only be possible if the requirement for ATP is met by glycolysis alone. As judged by the amount of lactate formed, the rate of glycolysis is enhanced during inhibition of mitochondrial protein synthesis in cultures in RPMI as well as in those in DME. From our data it follows that an enhanced glycolysis can compensate for the loss of ATP-generating capacity if the cells are cultured in DME, but not if they are cultured in RPMI. An exception to this general rule are P19 EC cells. Cultured in RPMI, these cells were able to perform about 5 cell divisions in the presence of DC before proliferation was arrested. There are no differences in the content of mitochondrial enzymes between P19 EC cells and the other 2 mouse cell lines used in this study (Van den Bogert et al., 1992). Despite the same activity of mitochondrial enzymes, such as cytochrome c oxidase and citrate synthase in the control cells, the different mouse cell lines appeared therefore to have different dependencies on mitochondrial functions. Since the oxidative phosphorylation capacity could be reduced to 2-39 of the normal value before proliferation of P19 EC cells was arrested, this implies that these cells can proliferate almost without mitochondrial ATP supply, even in RPMI. This might reflect a general trait of very early embryonic cells, namely, a large independency of the cells on mitochondrial function (Piko and Chase, 1973; Piko and Taylor, 1987). P19 EC cells responded to inhibition of mitochondrial protein synthesis when cultured in RPMI a s the other cell lines did when cultured in DME. This leads to the conclusion that cultured cells can do without mitochondrial ATP production if glycolysis can provide the ATP
required, either because the cells are grown in “rich” media (such as DME), which apparently reduces the need for ATP, or because the cells concerned have a n inherently low need for ATP (as might be the case in P19 EC cells). Nonetheless, even where glycolysis alone can satisfy the need for ATP, all cell lines stopped dividing in the absence of pyruvate when mitochondrial protein synthesis was continuously inhibited. This was always observed after 5-6 divisions of cultures grown in the presence of DC. Under these conditions, functional respiratory chains will hardly be present any more. As has been shown before, this will impair the synthesis of uridine (Gregoire et al., 1984).It has, however, also been shown that undialysed FCS, as used in our study, contains sufficient uridine to prevent the effects of lack of uridine synthesis (Karle et al., 1984). In line with this, we found that addition of extra uridine to the cultures did not prevent proliferation arrest. Another consequence of the absence of functional respiratory chains will be the accumulation of NADH inside the mitochondria. It is highly unlikely that this can be completely re-oxidised via glycolysis (Robinson e t al., 1990). Accumulation of NADH will result in impairment of several metabolic pathways inside the mitochondria because of feedback inhibition. This might be the reason why pyruvate is able to prevent proliferation arrest under these conditions. It has been shown for many cultured cell lines that glutamine, apart from being a n important energy source, is also indispensable as a precursor of for instance aspartate, which in turn is a n important precursor in the synthesis of several amino acids and ribonucleotides (McKeehan, 1982; Moreadith and Lehninger, 1984; Brand et al., 1986; Lanks et al., 1988). The formation of aspartate from glutamine will, however, be severely impaired if mitochondrial NADH levels are high. At least theoretically, it is con-
MITOCHONDRIA AND PROLIFERATION OF MAMMALIAN CELLS
0
3
6
3 .6
9
days o culturing in the
Fig. 3. Activity of mitochondrial enzymes, protein, and ATP content of Molt-4 cells cultured in the presence of DC under various conditions. The respective values are given as percentages of the control values, and represent the mean ofthe percentages found in at least 3 different experiments. A : protein content 11008: 118 mg/lOgcells, n = 250); :. ATP content ( 1 00%: 4.0nmol/lO" cells, n = 90); 'I: cytochrome c oxidase activity (100%: 26\10' cells, n = 250); 0:citrate synthase activity (1008:4.8/10" cells, n = 50); 0:number of cell divisions. A. Cells cultured in RPMl(25 mM glucose, 1mM pyruvate, 10% (20,). B. Cells cultured in DME (25 mM glucose, 1mM pyruvate, 5%'CO,). C. Cells cultured in DME (26 mM glucose, 1mM pyruvate, 10% CO,).
ceivable that aspartate is formed from pyruvate via either oxaloacetate or malate under these conditions. The former route requires pyruvate carboxylase and aspartate-oxaloacetate transaminase as enzymes and the latter malic enzyme. For both pathways, CO, is also required. ,411 3 enzymes are known to be present in cultured mammalian cells (Donelly and Schleffler, 1976). This explanation is supported by extensive studies on Chinese hamster ovary cell mutants without a functional respiratory chain. Here, proliferation remained possible when pyruvate or aspartate were added to the culture medium, and moreover these cells showed CO, dependency (Whitfield, 1985). At least for Molt4 cells, we also found a strong dependency on the C 0 2tension for growth. It is very likely that this dependency also exists for the other cell lines, since CO, will hardly be produced intracellularly if mitochondrial intermediairy metabolic pathways are inhibited. A CO, tension of 5% was apparently sufficient for the other cell lines; in M o b 4 cell cultures it had to be raised to 10% to allow proliferation to continue. This might be
637
related to the fact t h a t Molt-4 cells grow in suspension: the number of cells per culture in this case is substantially higher than in cultures of monolayers. In line with this assumption, Molt4 cells cultured for a prolonged time in the presence of DC could only be kept in a proliferating state by keeping the cells at low densities. Taken together, the results show that cultured mammalian cells are able t o survive without functional mitochondria if their ATP demand is reduced such that it can be supplied by glycolysis only and if pyruvate is present a s a n alternate precursor for compounds that are normally produced via mitochondrial metabolism. If these conditions have been fulfilled, the cells are apparently able to adjust their growth rate, size, and energy charge and remain proliferating. These conditions are highly unlikely to be met under more physiological conditions: in our previous work using several tumors as systems in vivo, inhibition of mitochondrial protein synthesis always resulted in proliferation arrest after 1-2 cell divisions (Van den Bogert et al., 1981, 1986a,b). Nonetheless, we feel the present data improve our insight in the relationship between mitochondria] function and proliferation of cultured cells. The data offer a number of explanations for apparently contradictory conclusions in the literature, e.g., on the question of whether or not cultured cells depend on mitochondrial ATP synthesis. In the majority of the cases, the answer will depend on the type of medium used. More important, our data are of value for the selection and maintenance of lines of cells t h a t are deficient with respect to 1 or more enzymes involved in oxidative phosphorylation, e.g., cell lines derived from patients with mitochondrial myopathies.
ACKNOWLEDGMENTS The members of theme 3 of the E.C. Slater Institute for Biochemical Research, University of Amsterdam are thanked for their cooperation and hospitality during many of the cell culture experiments. We also thank Drs. P.A. Bolhuis, N.H. Herzberg (Department of Neurology, University of Amsterdam1 and A.J. Meijer (E.C. Slater Institute for Biochemical Research) for their interest. Part of this study was made possible by a fellowship of C.V.d.B. of the Royal Netherlands Academy of Arts and Sciences, and a grant from "Het Prinses Beatrix Fonds," The Hague, The Netherlands.
LITERATURE CITED Borst, P., Ruttenberg, G.J.C.M., and Kroon, A.M. (1967) Mitochondrial DNA I. Preparation and properties of mitochondrial DNA from chick liver. Biochim. Biophys. Acta, 149t140-155. Brand, K., Leibold, W., Luppa, P., Schoerner, C . ,and Schulz, A. (1986) Metabolic alterations associated with proliferation of rnitogen-activated lymphocytes and of lymphoblastoid cell lines: Evaluation of glucose and glutamine metabolism. Immunobiol., 173:23-34. Donelly, M., and Schleff'ler, I.E. 11976)Energy metabolism in respiration-deficient and wild tvue Chinese hamster fibroblasts in culture. J. Cell. Physiol., 89:39-k%. Ilonofrio, J., Coleman, M.S., Hutton, cJ.J.,Daoud, A., Lampkin, B., and Dyminski, J . (1978) Overproduction of adenine deoxynucleosides and deoxynucleotides in adenosine deaminase deficiency with severe combined immunodeficiency disease. J. Clin. Invest., 62:884887. Gr(.goire, M., Morais, R., Quillam, M A . , and Gravel, D. (1984) On auxotrophy for pyrimidines of respiration-deficient chick embryo cells. Eur. J . Biochem., 142t49-55.
638
VAN DEN BOGERT ET AL.
Harris, M. (1980) Pyruvatc blocks cxpression of sensitivity to antimycin A and chloramphenicol. Som. Cell Genet., 6:699-708. Howell, N., and Lee, A. (1989) Sequence analysis of mouse mitochondrial chloramphenicol-resistant mutants. Som. Cell Genet., 25t237244. Howell, N., and Sager, R. (1979) Cytoplasmic genetics of mammalian cells: conditional sensitivity to mitochondrial inhibitors and isolation of new mutant phenotypes. Som. Cell Genet., FitS33-845. Karle, J.M., Anderson, L.W., and Cysyk, R.L. (1984) Effcct of plasma concentrations of uridine on pyrimidine biosynthesis in cultured L1210 cells. J . Biol. Chem., 259:67-72. King, M.P., and Attardi, G. (1988) Injection of mitochondria into human cells leads to a rapid replacement of the endogeneous mitochondrial DNA. Cell, 52t811-819. King, M.P., and Attardi, G. (1989)Human cells lacking mitochondrial DNA: 'depopulation with exogenous mitochondria by complementation. Science,246:500-503. Lanks, K.W.; and Li, P-W. !1988) End products of glucose and glutamine metabolism by cultured cell lines. J. Cell. Physiol., 235t151~ 155. Leezenberg, J.A., Wesseling, H., and Kroon, A.M. (1979)Possible cytostatic action of tetracyclines in the treatment of tumors of the nasopharynx and larynx. Eur. J . Clin. Pharmacol., 26237-241. McBurney, M., and Rogers, B.J. (1982) Isolation of male embryonal carcinoma cells and their chromosome replication patterns. Dev. Biol., 89503-508. McKeehan, W.L. (1982) Glycolysis, glutaminolysis and cell proliferation. Cell Biol. Tnt. Rep.. 6.535-651. Morais, R., and Giguere, L. (1979) On the adaptation of cultured chick embryo cells to growth in the presence of chloramphenicol. J. Cell. Physiol., 101:77-88. Morais, R., Gregoire, M., Jeanottc, L., and Gravel, D. 11980) Chick embryo cells rendered respiration deficient by chloramphenicol and ethidium bromide are auxotrophic for pyrimidines. Biochem. Biophys. Res. Comm., 94t71-77. Morais, R., Guertin, D., and Kornblatt, J.A. (1982) On the contribution of the mitochondrial genome to the growth of Chinese hamster embryo cells in culture. Can. J. Biochem., 60t290-294. Morais, R., Desjardins, P., Turmel, C., and Zinkewich-Pkotti, K. (19881 Development and characterization of continuous avian cell lines depleted of mitochondrial DNA. In Vitro Cell. Dev. Biol., 24,549658. Moreadith, R.W., and Lehninger, A.L. (1984) The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. J. Biol. Chem., 259t62154221. Mummery, C.L., Feyen, A,, Van den Brink, S., Molenaar, W.H., and De Laat, S.W. (1986) Establishment of a differentiated mesodermal line from P19EC cells expressing functional PDGF and EGF-receptors. Exp. Cell Res., 165329-242. Nelson, B.D. (1987) Biogenesis of mammalian mitochondria. In: Current Topics in Bioenergetics, Vol. 15. C.P. Lee; ed. Academic Press, San Diego, pp. 221-272. Peterson, G.L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem., 831346-356. Piko, L., and Chase, D.G. (1973) Role of the mitochondrial genome during early development in mice. Effects of ethidium bromide and chloramphenicol. J. Cell Biol., 58,357378.
Pik6, L., and Taylor, K.D. (1987)Amounts of mitochondrial DNA and abundance of some mitochondrial gene transcripts i n early mouse embryos. Dcv. Biol., 123336L374. Robinson, B.H., Glerum, D.M., Chow, W., Petrova-Benedict, R., Lightowlers, R., and Capaldi, R. (1990) The use of skin fibroblasts cultures in the detection of respiratory chain defects in patients with lacticacidemia. Pediatr. Res., 28t549-555. Srere, P.A. (1969)Citrate synthase. In: Methods in Enzymology, Vol. 13. J.M. Lowenstein, ed. Academic Press, London, pp. 3-26. Stanisz, J., Wice, B.M., and Kennell, D.E. 11983)Comparative energy metabolism in cultured heart muscle and Hela cells. J . Cell. Physiol., 115t320-330. Van den Bogert, C., Dontje, B.H.J., Wijbenga, J.J., and Kroon, A.M. (1981) Arrest of in vivo proliferation of Zajdela tumor cells by inhibition of mitochondrial protein synthesis. Cancer Res., 41.19431947. Van den Bogert, C., Lont, M., Mojet, M., and Kroon, A.M. (1983) Impairment of liver regeneration during inhibition of mitochondrial protein synthesis by oxytetracycline. Biochim. Biophys. Acta, 722r393-400. Van den Bogert, C., Dontje, B.H.J., Holtrop, M., Melis, T.E., Romijn, J.C. Van Dongen, J.W., and Kroon, A.M. (1986a) Arrest of the proliferation of renal and prostate carcinomas of human origin by inhibition of mitochondrial protein synthesis. Cancer Res., 46t32833289. Van den Bogert, C., Van Kernebeek, G., De Leij, L., and Kroon, A.M. (1986b)Inhibition of mitochondrial protein synthesis leads to proliferation arrest in the G1-phase ofthe cell cycle. Cancer Lett., 32r4151. Van den Bogert, C., Muus, P., Haanen, C., Pennings, A,, Melis, T.E., and Kroon, A.M. (1988)Mitochondrial biogenesis and mitochondrial activity during the progression of the cell cycle of human leukemic cells. Exp. Cell Res., 178:143-153. Van den Bogert, C., Pennings, A,, Dekker, H.L., Boezeman, J.H.M., Luciakova, K., and Sinjorgo, K.C.M. (1991) Quantification of mitochondria] proteins in cultured cells by immuno-flow cytometry. Biochim. Biophys. Acta, 1097r87-94. Van den Bogert, C., Dekker, H.L., Cornelissen, J.C., Van Kuilenburg, A.B.P., Bolhuis, P.A., and Muijsers, A.O. (1992) Isoforms of cytochrome c oxidase in tissues and cell lines of the mouse. Biochim. Biophys. Acta, 1099:11%122. Whitfield, C.D. (1985)Mitochondrial mutants. In: Molecular Cell Genetics. M.M. Gottesmann, ed. John Wiley & Sons, New York, pp. 545-689. Wicc, B.M., Reitzer, L.J., and Kennell, D. 119811 The continuous growth of vertebrate cells in the absence of sugars. J. Biol. Chem., 265t7812-7819. Zielke, H.R., Ozand, P.T., Tildon, J.T., Sevdalian, D.A., and Cornblath, M. (1976) Growth of human diploid fibroblasts in the absence of glucose utilization. Proc. Natl. Acad. Sci. USA, 73t4110-4114. Zielke, H.K., Ozand, P.T., Tildon, J.T., Sevdalian, D.A., and Cornblath, M. (1978) Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts. J . Cell. Physiol., 95.41-48. Zielke, H.R., Zielke, C.L., and Ozand, P.T. (1984) Glutamine: A major energy source for cultured mammalian cells. Federation Proc., 43t121-125.
