DENSITY-DEPENDENT SURVIVAL OF EHRLICHASCITES TUMOUR CELLS IN THE PRESENCE OFVARIOUS SUBSTRATES FOR ENERGY METABOLISM
E. ZAPOROWSKA-SIWIAK, M. MICHALIK, J. KAJSTURA ANDW.KOROHODADepartment of Cell Biology, Institute of Molecular Biology, Jagiellonian University, al.Mickiewicza 3, 31—120 Krakow, Poland
SUMMARY
We have found that Ehrlich ascites tumour (EAT) cells, deprived of any carbon source, andsuspended at a density of 2 X 10s cells/cm3, begin to die only after 12h of starvation, though it isknown that under these conditions they lose over 80% of their ATP within 30min. Moreover, wehave found that the viability of the cells incubated in the absence of any substrate for energymetabolism is strongly dependent on the density of the cell suspension, and can be significantlyimproved simply by increasing the suspension density. This prompted us to investigate the densitydependence of the maintenance of EAT cell viability in the presence of various substrates for energymetabolism and metabolic intermediates. It was found that:
(1) Glucose ensures 48 h viability of EAT cells irrespective of suspension density.(2) Fatty acids and pyruvate as sole carbon source do not improve EAT cell survival.(3) In the presence of glutamine as sole carbon source the EAT cell survival shows dependence
on cell-suspension density. At densities of 1-6 X 106 to 3-2 X 106 cells/cm3 the cell viability ismaintained at least as well as in the presence of glucose, but at low cell-suspension densitiesglutamine does not support cell viability.
(4) In the presence of glutamine, addition of 1 mM-inosine and 1 mM-uridine ensures high cellsurvival irrespective of the cell-suspension density.
(5) In the presence of inosine or uridine (10 mid) as sole carbon source, the EAT cell survival isthe same as in the presence of glucose and does not depend upon cell-suspension density. Guanosineis less effective, whereas adenosine has no effect at all on the maintenance of EAT cell viability for48 h.
(6) There is no correlation at all between EAT cell survival and the rate of lactic acid production.At a cell-suspension density of 1-6 X 106 cells/cm3 the cell survival is of the same order in thepresence of glutamine as in the presence of glucose, in spite of the fact that in the first case the rateof lactic acid production is more than 20 times lower.
(7) There is no correlation between the capacity of particular nucleosides to support EAT cellsurvival and their effects on glycolysis and oxygen consumption.
INTRODUCTION
It was reported that EAT cells and numerous other neoplastic cells, in contrast tonormal cells, are unable to maintain both their ATP levels and their viability inhexose-free media (Demetrakopoulos, Linn & Amos, 1978, 1982; Live & Kaminskas,1975). This feature of neoplastic cells was interpreted to be associated with the highrates of glycolysis usually observed in tumour cells (Demetrakopoulos et al. 1982).
Nevertheless, as shown by Live & Kaminskas (1975), EAT cells that under glucosestarvation had drastically decreased intracellular ATP and adenylate energy chargewere still able to increase their levels to the normal range if they were supplementedwith glucose after 5 h of starvation. In our preliminary experiments we observed thatEAT cells at a density of 2 X 105 cells/cm3 and deprived of any exogenous substratesfor energy metabolism remained viable over 12 h and only then began to die.Therefore, further studies are required to determine whether the depletion of cellularATP is really the sole and direct cause of cancer cell death upon deprivation ofhexoses. Zielke et al. (1976) and Zielke, Zielke & Ozand (1984) suggested that thecause of cell death in the absence of glucose was not related to its role as an energysource, but rather to its role as a substrate for anabolic reactions. The lack of a directrole for glycolysis in the maintenance of cancer cell survival is also suggested by dataindicating that several normal and tumour cell types can continue growth in theabsence of hexoses (Krause & Schneider, 1973; Zielke et al. 1976; Wice, Reitzer &Kennell, 1979) and that glutaminolysis can replace carbohydrate metabolism toprovide the energy required by the cells (Reitzer, Wice & Kennell, 1979; McKeehan,1982; Zielke et al. 1984). Moreover, Chan, Fung, Choy & Lee (1983) reported that,in vivo, ascites cells grow in ascitic fluid in which the glucose concentration is almosttoo low to be measured.
The study presented here has been designed to clarify the reasons for the divergencein data concerning the necessity of hexoses for the survival of cancer cells. It has beencarried out on Ehrlich ascites tumour cells and the emphasis was on determining theconditions influencing the survival of these cells when supplied with various sourcesof carbon.
MATERIALS AND METHODS
Cell cultureEAT cells were maintained in vivo in female Swiss albino mice (Camura Breeding Laboratories,
Krak6w, Poland), recovered and washed before in vitro culture, as described before (Cieilak &Korohoda, 1978). In vitro EAT cells were incubated in Leighton tubes at 37°C. The incubationmedium was Dulbecco's phosphate-buffered saline (PBS) supplemented with an appropriate carbonsource, 5% heat-inactivated dialysed calf serum, phenol red, lOOi.u./ml penicillin and 10^g/mlstreptomycin. The cell counts were done in a Burker haemocytometer. The viability was determinedby the Trypan Blue test. This test, based on an examination of the maintenance of cell plasmamembrane integrity as a permeability barrier, gives results that agree quantitatively with thoseobtained using the fluorescein diacetate test, based on the preservation of esterase activity(Szydtowska et al. 1978), recommended for determination of cell survival by Kemp, Meredith,Gamble & Frost (1983).
Lactate productionThe EAT cells were incubated in the appropriate medium for 6 h, the cells were then centrifuged
off, and the lactate was measured in the supernatant by the lactate dehydrogenase method.
Oxygen consumptionOwing to a continuous decline in the rate of oxygen consumption by EAT cells during the first
hours in vitro, cells were pre-incubated for 16 h at a density of 4 x 106 cells/cm3 in a medium
Density-dependent survival of EAT cells 77without any substrate for energy metabolism. Then a sample of cell suspension was withdrawn,washed and transferred to a respirometer chamber filled with the incubation medium containing nosubstrate for energy metabolism. The oxygen level was monitored with a Clark-type electrode (OP9343, Radelkis, Hungary) for 20 min, then the appropriate substrate was added and the record wascontinued for another 20 min.
ChemicalsAdenosine, glutamine, guanosine, inosine and uridine were obtained from Reanal, Hungary,
glucose and sodium pyruvate were from POCh, Poland. PBS and calf serum were from WSiS,Lublin, Poland.
RESULTS
In the initial experiment we determined the kinetics of the death of EAT cells whenkept at a suspension density of 2 X 105 cells/cm3 in Dulbecco's balanced salt solutionfree of any source of carbon and supplemented with dialysed calf serum, as describedin Materials and Methods. The data shown in Fig. 1 indicate that a significant percen-tage of EAT cells begin to die only after 14 h of carbon starvation, and after 28 h ofstarvation over 98 % of the cells are dead. In all our other series of experiments thepercentages of cells surviving after 48 h of incubation in tested media have beendetermined.
Fig. 2 shows the dependence of EAT cell survival upon cell-suspension density. Inthe presence of glucose the high viability of EAT cells was maintained at a cell-suspension density in the range from 0-2 X 106 to 1*6 X 106 cells/cm3. At the highercell densities lactic acid acidified the medium too strongly, whereas at lower cell
100
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tri-s^
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Fig. 1. Time dependence of EAT cell viability in the absence of any substrate for energymetabolism. Cells were incubated at a density of 2 X 10s cells/cm3. Vertical bars representstandard deviation.
78 E. Zapomwska-Swriak and others
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Fig. 2. Density-dependent viability of EAT cells after incubation for 48 h in the presenceof various carbon sources: ( • • ) no carbon source; (O O) 5-5mM-glucose;( • • ) 11 mM-glutamine. Vertical bars represent standard deviation.
densities the cell survival decreased. In the absence of any carbon source the cellsurvival gradually improved with an increase in the cell-suspension density and at12-8 X 106 cells/cm3 over 65% of cells survived after 48h of starvation. Thisobservation contradicts all those suggestions that postulated the necessity of hexosesand the maintenance of a high rate of glycolysis (see also Table 1) for survival of cancercells; and, consequently, it suggests that EAT cells can use substrates other thanhexoses to maintain high viability (e.g. products of serum proteolysis) and, or, thatcell death at low cell-suspension densities might result from causes other than aninadequate supply of substrates for energy metabolism and ATP regeneration.
To examine this idea, cell survival in the presence of various carbon sources, whichmight be postulated to be used for cell energy metabolism, has been determined.
Pyruvate, which stimulates respiration in chick embryo fibroblasts (Kajstura &Korohoda, 1983) and improves the survival and growth of those cells at low celldensities (Rein & Rubin, 1971), had no effect upon the density-dependent survivalof EAT cells in glucose-free medium. Sodium butyrate (5-5 mil) as well as linoleate(2'OmM) decreased rather than increased the percentage of EAT cells surviving for48h in glucose-free media (data not shown). However, when EAT cells were in-cubated in a glucose-free medium supplemented with glutamine (11 mM) the cellsurvival significantly improved and at the same time the dependence of EAT cellsurvival upon the cell-suspension density became more marked (Fig. 2). At a cell-suspension density of 10s cells/cm3 only 1-7 % of cells survived, whereas at a densityof 1 -6 X 106 cells/cm3 over 85 % of cells survived for 48 h after incubation in the same
Density-dependent survival of EAT cells 79
Table 1. Lactate production and oxygen consumption by EAT cells in the presence ofvarious substrates for energy metabolism and metabolic intermediates
medium. The density dependence of EAT cell survival in the presence of glutamineas a carbon source strongly suggests that the death of these cells at lower cell-suspension densities does not result solely from an inadequate supply of substrates forcellular energy metabolism and ATP regeneration reactions. From the results shownin Fig. 2 and the published data at least two other factors should be taken into accountwhen the survival of EAT cells in glucose-free media is analysed.
The absence of glucose in the medium can influence not only glycolysis, but alsothe pentose phosphate pathway and the pathways of synthesis of purine andpyrimidine nucleosides and nucleotides (Krause & Schneider, 1973; Zielke et al.1984). Hence not only might ATP be lost from glucose-starved cells but also otherintermediates of cellular metabolism. At high cell-suspension densities such inter-mediates leaking from the cells to the medium might be reutilized, ensuring themaintenance of cell viability. Following this line of reasoning, experiments weredesigned to ascertain whether or not nucleosides can influence the density-dependentsurvival of EAT cells in glucose-free media. Nucleosides have been chosen becausethey are taken up by cells much faster than the corresponding bases and pentoses andwere found to support the survival and growth of some cell types in tissue culture(Krause & Schneider, 1973; Wiceet al. 1981; Young & Jarvis, 1983).
Fig. 3 shows the effect of the addition of inosine (1 min) and, or, uridine (1 mM)to glucose-free, but glutamine-supplemented, medium on the cell-suspension densitydependence of 48 h survival of EAT cells. The addition of one or both of thesenucleosides improved the survival of EAT cells at low cell-suspension densities andhad no harmful effect on the survival of cells at high densities. Under these conditionsthe cell survival reached at least the same level as in glucose-containing medium. Theaddition of inosine alone improved the survival at low cell-suspension densities
80 E. Zapomwska-Siwiak and others
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Cell density (xll)-' cells/cm')
Fig. 3. Effect of nucleosides on the density-dependent viability of EAT cells in thepresence of glutamine (11 ITIM). ( • • ) No nucleosides added; (O O) 1 mM-inosine + lmM-uridine; (Q D) lmM-uridine; (A A) 1 niM-inosine. Viabilitywas determined after 48 h of incubation. Vertical bars represent standard deviation.
(2 X 104 to 10 X 104 cells/cm3), but decreased cell survival at a high cell-suspensiondensity, whereas the addition of uridine alone had a much less pronounced effect.
Fig. 4 presents the results of experiments on the effects of inosine and, or, uridine,added at lmM or lOmM-concentrations as sole carbon source to a glucose-freemedium, upon the density-dependent 48 h survival of EAT cells. Concentrations of1 mM, in particular when uridine and inosine were present together, significantlyimproved EAT cell survival.
When inosine and uridine were present at 5 mM concentration, or inosine alone waspresent at 10 mM, the EAT cell survival was equally as good as in the glucose-containing media, irrespective of cell-suspension density. Therefore, in thesubsequent experiments the effects of three purine nucleosides, i.e. inosine,guanosine and adenosine, were compared. Of these three nucleosides, inosine had thegreatest effect, guanosine improved cell survival markedly but less than inosine,whereas adenosine had no effect at all (Fig. 5).
To examine how the substrates tested influence the energy metabolism of EAT cellsincubated at a density of 1-6 X 106 cells/cm3, their effects upon lactate production andoxygen consumption were measured. The results are presented in Table 1. They showthat the pattern of cell energy metabolism strongly depends upon a particular type of
Density-dependent survival of EAT cells 81
KK) r
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Fig. 4. Effect of nucleosides on the density-dependent EAT cell viability in the absenceof other carbon sources. Cells were incubated for 48 h in the presence of: 1 mM-inosine( • • ) ; 1 mM-uridine (A A); 1 mM-inosine + 1 mM-uridine ( ^ •);10mM-inosine ( • • ) ; 10mM-uridine (A A); 5 mM-inosine + S mM-uridine
O)- (9 # ) Control without nucleosides.
carbon source and that there is a lack of any direct correlation between cell survivaland the rates of lactic acid production and oxygen consumption.
Though the 48 h survival of EAT cells in the presence of glutamine was the sameas in the presence of glucose (at this cell-suspension density), in the first case glycoly-tic metabolism dominated, whereas in the second respiration dominated and lacticacid production was more than 20 times lower.
A similar order of cell survival was observed in the presence of inosine, which hadno effect upon respiration and stimulated lactic acid production, but at a level seventimes lower than glucose. Uridine, which also supported cell survival, stimulatedglycolysis 2-3 times less than inosine and 16 times less than glucose. However,guanosine, which stimulated lactic acid production even more strongly than inosine,supported EAT cell viability less effectively than inosine or uridine.
DISCUSSION
The experiments presented here were designed to examine whether: (1) hexosescan be replaced by other substrates for energy metabolism to ensure maintenance of
82 E. Zaporowska-Siwiak and others
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Fig. 5. Effect of purine nucleosides on the density-dependent viability of EAT cells in theabsence of other carbon sources. Cells were incubated for 48 h in the presence of: 10 mM-inosine (O O); 10mM-guanosine ( • • ) and lOmM-adenosine (A A) .( • • ) Control without nucleosides. Vertical bars represent standard deviation.
EAT cell viability; (2) metabolic intermediates other than those directly involved incell energy metabolism can prevent EAT cell death in the absence of glucose.
Our preliminary experiments have shown that in the absence of any exogenoussubstrates for energy metabolism the survival of EAT cells is dependent on cell-suspension density. At high cell-suspension density the cell survival was found to besignificantly improved.
The lack of improvement of EAT cell survival in the presence of pyruvate and fattyacids in glucose-free media can be explained on the basis of published data. Villalobo& Lehninger (1980) reported that pyruvate only slightly stimulates respiration ofmitochondria isolated from EAT cells, and Paradies et al. (1983) showed that inmitochondria isolated from EAT cells the activity of the pyruvate translocator is muchlower than in mitochondria isolated from rat liver cells. According to Busch (1962),in Jensen sarcoma and Walker 256 tumour cells also, in contrast to normal cells,pyruvate is not metabolized via the Krebs cycle. Similarly, Sumbilla et al. (1981)showed that human diploid fibroblasts cannot use the oxidation of fatty acids andketone bodies for their energy requirements, and Stanisz, Wice & Kennell (1983)reported that HeLa cells derive little or no energy from oxidation of fatty acids.
Density-dependent survival of EAT cells 83
There is now substantial experimental evidence that glutamine can be the majorsubstrate for respiration in tumour cells (Reitzer et al. 1979; Moreadith & Lehninger,1984). We observed that replacement of glucose by glutamine greatly improved EATcell survival if no other substrates for energy metabolism were present. When the cellswere incubated at 8 X 10s to 32 X 105 cells/cm3 cell-suspension density, the cell sur-vival was similar to that in the presence of glucose. Apparently, an increase in cellrespiration caused by glutamine (Table 1) fully compensated the 20-fold decrease inthe rate of glycolysis. This strongly supports the conclusions of Krause & Schneider(1973), Reitzer et al. (1979), Zielke et al. (1976, 1984), Franchi, Silvestre &Pouyssegur (1981), Wice et al. (1981) and Kajstura & Korohoda (1983), thatglycolysis itself has no direct significance for the maintenance of normal and neoplasticcell survival and growth. Moreover, the observation that at high cell densitiesglutamine can support cancer cell viability in the absence of, or with minimal,glycolysis seems to explain why numerous attempts to inhibit cancer growth in vivoby various inhibitors of glycolysis have been unsuccessful (cf. Busch, 1962; Tannock,Guttman & Rauth, 1983).
The lack of beneficial effects of glutamine on the survival of EAT cells incubatedat densities lower than 2 X 105 cells/cm3 suggests that under these conditions celldeath results from causes other than an inadequate supply of substrates for energymetabolism.
The cell-density dependency of EAT cell survival in the presence of glutamine assole carbon source shows that the problem of cancer cell death in the absence ofhexoses cannot be reduced to the problem of energy conservation reactions. In theabsence of hexoses not only is glycolysis impeded but also the pentose pathway, as wellas the pathways of synthesis of purine and pirymidine nucleotides. The results presen-ted in this paper show that inosine and uridine significantly improve EAT cell survivalin the absence of other carbon sources. Surprisingly, adenosine has no effect, whereasguanosine improves cell survival less effectively than inosine. What is more, thesedifferences in the capacity of particular nucleosides to support EAT cell survival arenot correlated with their effects on lactate production and oxygen consumption.
The particularly great capacity of inosine to support cell survival and growth in theabsence of glucose, but in the presence of all the amino acids in Eagle's MEM, havebeen observed already in the case of EAT cells (Krause & Schneider, 1973) and pigerythrocytes (Jarvis et al. 1980). As we observed, lOmM-inosine as sole carbonsource, even in the absence of amino acids, can ensure the maintenance of a highviability for EAT cells. What is more, the viability in the presence of 10 mM-inosineas the sole carbon source is not density dependent. Uridine, which was reported toensure the growth of HeLa cells in the presence of amino acids in glucose-free media(Wice et al. 1981), was observed to improve the survival of EAT cells, even as the solecarbon source. Again, in this case the cell survival was not density-dependent. Incontrast, adenosine had no effect on EAT cell survival in the absence of substrates forenergy metabolism. In virally transformed mammalian cells a lack of adenosinedeaminase was reported (Shipman & Drach, 1978). If this enzyme is also absent inEAT cells, this could explain the difference between the ability of adenosine and
84 E. Zaporowska-Simiak and others
inosine or guanosine to support the viability of these cells.The results presented in Table 1 show the lack of any direct dependence of EAT
cell survival upon the rate of glycolysis, and suggest that for EAT cells to maintaintheir viability they require only a minimal level of ATP regeneration reactions, irres-pective of their nature. What is more, marked differences in EAT cell survival obser-ved in the presence of four tested nucleosides suggest that ribose itself is not sufficientfor the maintenance of cell viability.
The results presented here indicate the significance of cell-suspension density as aparameter that should be taken into account in any study concerning cancer cellmetabolism, survival and growth. The plasma membranes of cancer cells are moreleaky than those of normal cells (Weiss, 1967). Because of this, these cells probablylose more metabolic intermediates than normal cells, modifying their local environ-ment more strongly. The leakiness of cancer cells to metabolic intermediates, ratherthan their doubtful dependence on glycolysis, might be the reason for their higherthan normal sensitivity to depletion of substrates for energy metabolism.
This work was supported by a grant II.1.3.6. from the Polish Academy of Sciences.
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(Received 21 December 1984 -Accepted 19 February 1985)
