ISSN 00062979, Biochemistry (Moscow), 2013, Vol. 78, No. 8, pp. 904908. © Pleiades Publishing, Ltd., 2013. Original Russian Text © E. I. Leznev, I. I. Popova, V. P. Lavrovskaja, Y. V. Evtodienko, 2013, published in Biokhimiya, 2013, Vol. 78, No. 8, pp. 11511157.
Comparison of Oxygen Consumption Rates in Minimally Transformed BALB/3T3 and VirusTransformed 3T3BSV40 Cells E. I. Leznev1,2*, I. I. Popova1, V. P. Lavrovskaja1,2, and Y. V. Evtodienko3 1
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, 142290 Pushchino, Moscow Region, Russia; fax: +7 (4967) 330553; Email: [email protected]; [email protected] 2 Pushchino State Natural Science Institute, pr. Nauki 3, 142290 Pushchino, Moscow Region, Russia; fax: +7 (4967) 732711; Email: [email protected] 3 Evik Diagnostics, Ottawa, Canada; Email: [email protected] Received December 13, 2012 Revision received February 11, 2013 Abstract—In the recent years, bioenergetics of tumor cells and particularly cell respiration have been attracting great atten tion because of the involvement of mitochondria in apoptosis and growing evidence of the possibility to diagnose and treat cancer by affecting the system of oxidative phosphorylation in mitochondria. In the present work, a comparative study of oxygen consumption in 3T3BSV40 cells transformed with oncovirus SV40 and parental BALB/3T3 cells was conducted. Such fractions of oxygen consumption as “phosphorylating” respiration coupled to ATP synthesis, “free” respiration not coupled to ATP synthesis, and “reserve” or hidden respiration observed in the presence of protonophore were determined. Maximal respiration was shown to be only slightly decreased in 3T3BSV40 cells as compared to BALB/3T3. However, in the case of certain fractions of cellular respiration, the changes were significant. “Phosphorylating” respiration was found to be reduced to 54% and “reserve” respiration, on the contrary, increased up to 160% in virustransformed 3T3BSV40 cells. The low rate of “phosphorylating” respiration and high “reserve” respiration indicate that under normal incubation conditions the larger part of mitochondrial respiratory chains of the virustransformed cells is in the resting state (i.e. there is no electron transfer to oxygen). The high “reserve” respiration is suggested to play an important role in preventing apop tosis of 3T3BSV40 cells. DOI: 10.1134/S0006297913080063 Key words: BALB/3T3 and 3T3BSV403T3 cell cultures, respirometry, cellular respiration, inhibitors of oxidative phospho rylation, tumor cells, programmed cell death
We have recently witnessed growing interest in stud ies of bioenergetics of cells with different levels of trans formation due to the possibility of tumor therapy based on influencing the systems of glycolytic and oxidative phosphorylation [15]. Many studies have focused on clarifying the reasons for the relatively high glycolysis and decreased respiration in tumor cells – phenomena first discovered by Warburg [6]. It has been established that the high level of glycolysis is associated with increased expression of a number of genes, this fact leading to the Abbreviations: MPTP, mitochondrial permeability transition pores; MRC, mitochondrial respiratory chains; PCD, pro grammed cell death; RC, respiratory control coefficients; RCADP, control of respiration by the system of ATP synthesis from ADP and phosphate; RCPMF, control of respiration by proton motive force. * To whom correspondence should be addressed.
activation of glucose transport and its utilization in the cells. This ensures efficient energy supply of tumor cells, particularly under hypoxic conditions [4]. As for the decrease in respiration level and its importance for tumor cells, this issue still remains controversial. Relatively low oxygen consumption and reduced activity of certain mitochondrial enzymes have been found in some trans formed cells, but low oxygen consumption has been also shown to be not a compulsory feature of tumor cells [1, 2, 4]. Study of oxygen consumption in mitochondria of tumor cells is of considerable interest due to the fact that mitochondria are responsible for the initial stages of pro grammed cell death (PCD), in particular for the develop ment of apoptosis and the release of apoptosisinducing factors into the cytosol [7, 8]. Suppression of PCD is now regarded as one of the main reasons for the high level of survival of tumor cells [8, 9].
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RESPIRATION OF 3T3 CELLS Some inconsistency in the data on bioenergetics of tumor cells is due to the fact that in a number of studies their respiration was compared to glycolysis only on a sin gle cell model, while in other cases, including the studies by Warburg, it is bioenergetics of tumor and normal cells of different origin that was the object of research. In this work, we compare respiration of homologous BALB/3T3 cells. We used a standard cell line of parental BALB/3T3 fibroblasts and a standard culture of the same cells infect ed with oncogenic SV40 virus as the objects for our study. Different versions of 3T3 cell lines are common materials for a wide range of studies, particularly for studies of virusinduced cell transformation. One of the reasons is that 3T3 cells cultures, depending on their age, have properties of both primary (less than 10 passages) and minimally transformed continuous cultures (passage number higher than 1012). Different 3T3 cell lines were first obtained and studied by Aaronson and Todaro; the same authors showed the possibility of their transforma tion by SV40 virus [10]. Morphokinetics and proliferation of various 3T3 cell lines were also studied and described by Russian researchers [11, 12]. Because oxygen consumption in mitochondria is not only connected to ATP synthesis, but also provides a number of other functions, in this work we studied partial reactions of oxygen consumption by BALB/3T3 and 3T3BSV40 cells. Several respiration fractions have been investigated: “phosphorylating” respiration coupled to ATP synthesis, “free” respiration not coupled to ATP synthesis, and “reserve” or hidden respiration induced by exogenous protonophore. Such a study is relevant as it allows estimating the changes in respiration fractions responsible for different mitochondrial functions in virus transformed and other tumor cells.
MATERIALS AND METHODS Cell cultures. We used mouse embryonic fibroblasts of BALB/3T3 line, clone A31 (hereinafter BALB/3T3) and the same fibroblasts transformed with SV40 virus, 3T3BSV40 cell line. The cultures were certified and obtained from the Russian Collection of Cell Cultures (Institute of Cytology of the Russian Academy of Sciences, St. Petersburg). Conditions for cell cultivation. The cells were cul tured according to standard procedure [13] and passport data for BALB/3T3 and 3T3BSV40. DMEM medium with glutamine and 4.5 g/liter of glucose (MP Biomedical, USA) was used for cell cultivation and incu bation. The following components were added to this medium: 10% fetal bovine serum (HyClone, USA), 1 g/liter sodium bicarbonate, 20 mM Hepes (Sigma, USA), and 80 µg/ml gentamicin (JSC Biokhimik, Russia). Cells were cultured in vials (25 cm2; Greiner, Germany) at 37°C. BALB/3T3 cells were seeded at BIOCHEMISTRY (Moscow) Vol. 78 No. 8 2013
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300,000400,000 cells per vial with 5 ml of medium and were removed after 34 days using trypsinEDTA solution (SPE PanEco). 3T3BSV40 cells were seeded at 400,000 500,000 cells per vial with 5 ml of medium and were removed after 34 days using a solution of 0.25% trypsin and 0.02% Versene (ratio 1 : 3). After removal, the cells were suspended in DMEM medium with the aforemen tioned additives (106 cells per ml of incubation medium). The number of cells was counted in a Goryaev chamber after trypan blue staining. The cells obtained in the labo ratory after 1220 passages were used for the experiments. Oxygen consumption by cells was studied using an Oxygraph2k device (OROBOROS Instruments, Austria) according to the recommended protocol [14, 15]. The Oxygraph2k device was used in standard configuration with two chambers of 2 ml each. Agitator speed was 750 rpm; temperature of the incubation medium was 37°C. The DatLab4 program was used for data collection and analysis. Cell incubation conditions. Cellular respiration was determined in cells incubated in DMEM medium of the abovedescribed composition. Cell concentration in each chamber was ≈106 cells per ml of the incubation medium. Oligomycin (Sigma) was used as an inhibitor of ATP synthesis, and carbonyl cyanide 4(trifluorometh oxy)phenylhydrazone (FCCP; Sigma) was used as a protonophoric uncoupler of oxidative phosphorylation. Oxygen consumption rate was measured in the absence of inhibitors of oxidative phosphorylation and in the pres ence of oligomycin (1 µg/ml of medium) and FCCP (2.5 µM) added to the incubation medium. Concentrations of FCCP and oligomycin were chosen on the basis of titration of respiration by these inhibitors. Maximal activation of respiration (100%) in 3T3BSV40 cells and 9095% activation of respiration in BALB/3T3 cells were observed at the selected FCCP concentration. In the case of selected oligomycin concentration, maxi mal inhibition of respiration was observed in both BALB/3T3 and 3T3BSV40 cells. Statistical analysis of experimental results. The Graph Pad Prisma 4 program was used for statistical analysis of the data. The samples were compared using the nonpara metric Mann–Whitney Utest. The differences were con sidered reliable at p < 0.05 (*). The tables show the mean values ± standard error of the means (the latter were cal culated from the data of 68 experiments for all the param eters of cellular respiration presented in this work). Determination and calculation of partial reactions of cellular respiration. The following partial rates of oxygen consumption by cells were measured and calculated: Vnorm – “normal respiration” or the base rate of oxygen consumption by cells under normal incubation condi tions in the absence of inhibitors and uncouplers of oxidative phosphorylation; Vfree – “free respiration” or the rate of oxygen consumption by cells measured in the presence of oligomycin, the inhibitor of mitochondrial
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FoF1ATP synthase; Vphosph – “phosphorylating respira tion” or oxygen consumption coupled to ATP synthesis carried out by mitochondrial FoF1ATP synthase, calcu lated as the difference between Vnorm and Vfree; Vmax – “maximal respiration” or the maximum possible rate of oxygen consumption by cells, this rate being determined in the presence of FCCP, the protonophore and uncou pler of oxidative phosphorylation; Vres – “reserve respira tion” or the rate of oxygen consumption calculated as the difference between Vmax and Vnorm. Glucose consumption was determined by using the test system AccutrendPlus (Roche Diagnostics). Glucose consumption was shown to be 1.2 and 5.5 nmol/min per 106 cells for BALB/3T3 and 3T3BSV40, respectively, when cells were cultured under aerobic conditions in the absence of inhibitors of oxidative phosphorylation.
RESULTS Partial respiration reactions of minimally transformed BALB/3T3 cells and virustransformed 3T3BSV40 cells. In this work, we comparatively studied the partial reactions of oxygen consumption by BALB/3T3 and 3T3BSV40 cells. We determined the rate of oxygen consumption by the cells under normal conditions in the incubation medi um, in the presence of oligomycin inhibiting ATP synthe sis, and in the presence of a saturating concentration of the protonophore FCCP. The data are shown in Table 1. Baseline oxygen consumption by cells under normal conditions, Vnorm, provides energy for ATP synthesis and other processes in mitochondria that require energy. The Vnorm value was shown to be reliably lowered in 3T3B SV40 cells (≈30%) when compared to minimally trans formed BALB/3T3 cells. “Free” oxygen consumption, i.e. not coupled to ATP synthesis in mitochondrial respi ratory chain (MRC), Vfree, was determined when FoF1 ATP synthase was blocked by oligomycin. The released energy is used for ion transport, including futile ion trans port, which produces heat. Vfree was shown to be slightly raised in 3T3BSV40 cells. “Phosphorylating” respiration provides energy for ATP synthesis in MRC. Vphosph was shown to be reduced almost twofold in 3T3SV40 cells
compared to BALB/3T3 cells. The maximal possible rate of oxygen consumption, Vmax, was determined in the presence of a saturating concentration of the protono phore FCCP. The averaged data for Vmax showed this parameter to be reduced by 17% in 3T3BSV40 cells. The fraction of respiration activated by FCCP, which we call “reserve respiration”, Vres, was also determined. This res piration remains “hidden” or “reserved” under normal conditions of cell incubation in the absence of exogenous protonophore. Vres was found to be reliably increased by almost 1.6fold in 3T3BSV40 cells. Functional analysis of partial reactions of oxygen con sumption in minimally transformed and virustransformed cultures of 3T3 cells. To assess the functional state of mitochondria in the cells, characteristics such as coeffi cients of “respiratory control”, i.e. control of respiration by the system of ATP synthesis from ADP and phosphate (RCADP) and control of respiration by the proton motive force (RCPMF), were calculated. The results are presented in Table 2. Respiratory control coefficient, RCADP, is widely used in bioenergetics in experiments on isolated mito chondria; it is calculated as the ratio of oxygen consump tion rates in the presence and in the absence of ADP or before and after inhibition of ATP synthesis by oligomycin. RCADP characterizes the degree of coupling between respiration and phosphorylation. As presented in Table 2, the traditional way of calculation (RCADP = Vnorm/Vfree) shows a relatively high value of respiratory control in BALB/3T3 cells, RCADP = 5.6. RCADP 4 is considered high in isolated mitochondria of normal cells. In the case of 3T3BSV40 cells, RCADP coefficient is reduced approximately twofold, this indicating a low degree of coupling between respiration and phosphoryla tion, i.e. in this case a significantly smaller portion of oxy gen consumed by cells is used for ATP synthesis. Control of respiration by proton motive force (RCPMF) is another parameter showing significant differ ences in the regulation of energy supply systems in BALB/3T3 and 3T3BSV40 cells. This value was calcu lated as the level of activation of the basic respiration by FCCP. This protonophore increases the leakage of H+ ions and decreases the proton motive force on the inner
Table 1. Partial rates of oxygen consumption by BALB/3T3 and 3T3BSV40 cells under different incubation condi tions (n = 68) Rate of oxygen consumption by cells, pmol/s per 106 cells Cells Vnorm
Vfree
Vphosph
Vmax
Vres
BALB/3T3
45.7 ± 9.7
9.8 ± 2.7
35.9 ± 10.1
58.7 ± 10.5
12.9 ± 3.3
3T3BSV40
30.8 ± 9.4*
11.5 ± 3.2
19.3 ± 7.1**
48.9 ± 8.8
20.4 ± 4.3*
Note: Statistically significant differences between the respiration in 3T3BSV40 and BALB/3T3 cells are indicated: * p < 0.05; ** p < 0.01.
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Table 2. Respiratory control coefficients in BALB/3T3 and 3T3BSV40 cells under different incubation conditions (n = 68) Respiratory control coefficients in cells Cells RCADP (Vnorm/Vfree)
Ratio Vphosph/Vfree
RCPMF (Vmax/Vnorm)
Ratio Vres/Vnorm
BALB/3T3
5.59 ± 1.51
4.59 ± 1.51
1.29 ± 0.06
0.29 ± 0.06
3T3BSV40
2.68 ± 0.39**
1.58 ± 0.43**
1.78 ± 0.23**
0.78 ± 0.23**
Changes in RC coefficients in 3T3BSV40*
48%
34%
138%
273%
* RC values in BALB/3T3 cells were assumed to be 100%. ** Statistically significant differences between the respiration in 3T3BSV40 and BALB/3T3 cells are indicated: p < 0.01.
mitochondrial membrane. The protonophore only slight ly activates respiration in BALB/3T3 cells (RCPMF = 1.3). However, in 3T3BSV40 cells it caused a significantly higher activation of respiration (RCPMF = 1.8). The revealed differences in RCADP and RCPMF values are well supported and are much more significant when individual components of respiration are analyzed, Vphosph instead of Vnorm and Vres instead of Vmax, respectively. As shown in Table 2, in this case the Vphosph/Vfree value is low ered threefold, and Vres/Vnorm value is increased almost threefold in 3T3BSV40 cells. Ratio between “phosphorylating”, “free”, and “reserve” oxygen consumption by BALB/3T3 cells and virustransformed 3T3BSV40 cells. As shown above in Table 1, the maximal rates of oxygen consumption in the presence of protonophore, Vmax, have similar values in BALB/3T3 and 3T3BSV40 cells. These data suggest that the potential ability of mitochondria to transfer electrons along the respiratory chain is not significantly changed in 3T3BSV40 when compared to BALB/3T3 cells. As Vmax is composed of three main components, “phosphorylat ing”, “free”, and “reserve” respiration, contribution of these three components in the maximal possible respira tion was estimated. Normalized values of Vphosph, Vfree, and Vres were calculated in each experiment, and average val ues were calculated based on these data. Normalized Vphosph and Vres values were shown to be strongly altered in 3T3BSV40 cells. In the case of BALB/3T3 cells, Vphosph constitutes 63% of Vmax, while in 3T3BSV40 cells this component is only 36% of Vmax. Due to restriction of Vphosph, mitochondrial respiratory potential in 3T3BSV40 cells is not fully realized under normal conditions. This observation is consistent with the fact that the protonophoreactivated fraction of respiration, Vres, con stitutes about 20% of Vmax in BALB/3T3 cells, while in the case of 3T3BSV40 cells this fraction is substantially increased, up to 41% of Vmax. “Free” respiration, Vfree, changed only slightly; it was shown to be 17 and 23% of Vmax in BALB/3T3 and 3T3BSV40 cells, respectively. BIOCHEMISTRY (Moscow) Vol. 78 No. 8 2013
DISCUSSION Partial reactions of cellular respiration of 3T3BSV40 cells transformed by Simian virus 40 and the parental BALB/3T3 cells have been comparatively studied. SV40 is generally accepted to be an oncogenic virus capable of inducing malignant transformation of different types of human and animal cells, including 3T3 cells [1618]. Tumor transformation is shown to occur due to the fact that SV40 T antigen binds tumor suppressor proteins such as pRb and p53 in host cells, these proteins being the key regulators of cell proliferation involved in arrest of the cell cycle and induction of PCD [1, 18, 19]. 3T3 cells infected by SV40 virus show signs of tumor cells: accelerated divi sion, decreased cell death, lower dependence of cell growth on the presence of serum in the cultivation medium, loss of contact inhibition of growth, increased glucose utilization and lactate production [2023]. In this connection, the approach used in this work to study partial reactions of res piration and the resulting data may be useful for under standing the role of cellular respiration not only in virus transformed cells, but also in other types of tumor cells. We observed a relatively small but significant decrease in basic respiration (Vnorm) in 3T3BSV40 cells. A similar decrease in basic respiration in 3T3 cells infect ed with oncogenic HRAS virus was described by Yang et al. [24]. We found a significant decrease in phosphorylat ing respiration (Vphosph) in 3T3BSV40 compared to BALB/3T3. Since the rate of phosphorylating respiration in 3T3BSV40 cells is less than the half of the maximal possible respiration rate, we can assume that electron transfer along the mitochondrial respiratory chains (MRC) is not the cause and limiting stage of the low Vphosph. There seem to be two possible reasons for the low Vphosph in 3T3BSV40 cells: a wellknown for tumor cells suppression of respiration by increased glycolysis as a result of competition for ADP and Pi (the Crabtree effect [25]) and reduced activity of mitochondrial FoF1ATP synthase caused by decreased expression of its βsubunit
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[26]. This conclusion is supported by the observation that under our experimental conditions glucose consumption was significantly increased in 3T3BSV40 cells. The high reserve respiration (Vres) found in this study is another principal difference of 3T3BSV40 cells. Due to the low rate of Vphosph, a significant part of the MRC does not transfer electrons to oxygen, being in a quiescent or reserve state (when 3T3BSV40 cells are incubated under normal conditions). In the absence of exogenous protonophore, arrest of electron transfer in reserve MRC should lead to an increased reduction level of carriers of electrons and hydrogen, such as NADH, NADPH, QH2, etc. Significantly increased NADH/NAD ratio in 3T3 SV40 cells, 34 times higher than in parental 3T3 cells, was shown by Schwartz et al. [27]. In experiments with isolated mitochondria and mitochondria in situ in cultured cells, MPTP opening and release of apoptosisinducing factors from mitochondria were shown to be coupled to the reduction of membrane potential and NADH oxidation [3, 7, 2831]. Hence, it seems reasonable to suggest that high “reserve respira tion” of 3T3SV40 cells found in this study may prevent MPTP opening, and thus this phenomenon cannot be viewed as a simple physiologically insignificant conse quence of reduced “phosphorylating” respiration; it rather plays a significant role in increased survival of virustransformed cells. It should be noted that the con cept of “reserve respiration” used in this work is analo gous to the concept of “respiratory capacity” (the differ ence between the maximal and base respiration) suggest ed and successfully applied by Nicholls et al. [32, 33] to estimate the survival of nerve cells under conditions of maximal physiological stimulus and in neuropathologies. The approach to the study of partial reactions of oxy gen consumption discussed in this paper seems to be quite useful for studies of bioenergetics of other transformed and tumor cells. Increased “reserve respiration” found in 3T3SV40 cells can be regarded as one of the targets for anticancer drugs. In this connection, it seems interesting to note that Chen et al. [33] reported that a number of nat ural anticancer compounds are currently undergoing clin ical trials, these drugs being aimed at correction of mito chondrial functions, including several compounds capable of decreasing mitochondrial membrane potential. REFERENCES 1. 2. 3. 4.
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5. Ramsay, E. E., Hogg, P. J., and Dilda, P. J. (2011) Pharm. Res., 28, 27312744. 6. Warburg, O. (1956) Science, 123, 309314. 7. Kroemer, G. (1999) Biochem. Soc. Symp., 66, 115. 8. Ouyang, L., Shi, Z., Zhao, S., Wang, F. T., Zhou, T. T., Liu, B., and Bao, J. K. (2012) Cell Prolif., 45, 487498. 9. Ferreira, C. G., Epping, M., Kruyt, F. A., and Giaccone, G. (2002) Clin. Cancer Res., 8, 20242034. 10. Aaronson, S. A., and Todaro, G. E. (1968) J. Cell. Physiol., 72, 141148. 11. Vasiliev, Yu. M., and Gelfand, I. M. (1981) Interaction of Normal and Neoplastic Cells with the Medium. Problems of Developmental Biology [in Russian], Nauka, Moscow. 12. Barkan, R. S., and Nikolsky, N. N. (1985) Tsitologiya, 27, 526. 13. Pinaev, G. P. (1988) Methods of Cell Cultivation [in Russian], Nauka, Leningrad. 14. Ashley, N., Garedev, A., Troppmair, J., Boushel, R., and Gneiger, E. (2005) Mitochondr. Physiol. Network, 10, 5557. 15. Pesta, D., and Gnaiger, E. (2012) Mitochondr. Bioenerg.: Methods Mol. Biol., 810, 2558. 16. Regis, A., Vilchez, S., and Butel, J. (2004) Clin. Microbiol. Rev., 17, 495508. 17. Geoffrey, M. C. (2000) The Cell, 2nd Edn., A Molecular Approach, Chap. 15, Tumor Viruses, Boston University, Sunderland (MA): Sinauer Associates. 18. Moens, U., Van Ghelue, M., and Johannessen, M. (2007) Cell Mol. Life Sci., 64, 16561678. 19. Ahuja, D., SaenzRobles, M. T., and Pipas, J. M. (2005) Oncogene, 24, 77297745. 20. Carrino, D., and Gershman, H. (1977) Proc. Natl. Acad. Sci. USA, 74, 38743878. 21. Elvin, P., and Evans, C. W. (1982) Eur. J. Cancer. Clin. Oncol., 18, 669675. 22. Colby, C., and Romano, A. H. (1975) J. Cell Physiol., 85, 1523. 23. Diamond, I., Legg, A., Schneider, J. A., and Rozengurt, E. (1978) J. Biol. Chem., 253, 866871. 24. Yang, D., Wang, M. T., Tang, Y., Chen, Y., Jiang, H., Jones, T. T., Rao, K., Brewer, G. J., Singh, K. K., and Nie, D. (2010) Cancer Biol. Ther., 9, 122133. 25. RodriguezEnriquez, S., Juarez, O., RodriguezZavala, J. S., and MorenoSanchez, R. (2001) Eur. J. Biochem., 268, 25122519. 26. Willers, I. M., and Cuezva, J. M. (2011) Biochim. Biophys. Acta, 1807, 543551. 27. Joan, P., Schwartz, J. V., Passonneau, G., Johnson, S., and Pastan, I. (1974) J. Biol. Chem., 249, 41384143. 28. Evtodienko, Y. V., Teplova, V. V., Azarashvily, T. S., Kudin, A., Prusakova, O., Virtanen, I., and Saris, N. E. (1999) Mol. Cell Biochem., 194, 251256. 29. Ly, J. D., Grubb, D. R., and Lawen, A. (2003) Apoptosis, 8, 115128. 30. Lemasters, J. J., Qian, T., He, L., Kim, J. S., Elmore, S. P., Cascio, W. E., and Brenner, D. A. (2002) Antioxid. Redox Signal., 4, 769781. 31. Norberg, E., Gogvadze, V., Ott, M., Horn, M., Uhlen, P., Orrenius, S., and Zhivotovsky, B. (2008) Cell Death Differ., 15, 18571864. 32. Brand, M. D., and Nicholls, D. G. (2011) Biochem. J., 435, 297312. 33. Choi, S. W., Gerencser, A. A., and Nicholls, D. G. (2009) J. Neurochem., 109, 11791191. 34. Chen, G., Wang, F., Trachootham, D., and Huang, P. (2010) Mitochondrion, 10, 614625.
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Comparison of Oxygen Consumption Rates in Minimally Transformed BALB/3T3 and VirusTransformed 3T3BSV40 Cells E. I. Leznev1,2*, I. I. Popova1, V. P. Lavrovskaja1,2, and Y. V. Evtodienko3 1
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, 142290 Pushchino, Moscow Region, Russia; fax: +7 (4967) 330553; Email: [email protected]; [email protected] 2 Pushchino State Natural Science Institute, pr. Nauki 3, 142290 Pushchino, Moscow Region, Russia; fax: +7 (4967) 732711; Email: [email protected] 3 Evik Diagnostics, Ottawa, Canada; Email: [email protected] Received December 13, 2012 Revision received February 11, 2013 Abstract—In the recent years, bioenergetics of tumor cells and particularly cell respiration have been attracting great atten tion because of the involvement of mitochondria in apoptosis and growing evidence of the possibility to diagnose and treat cancer by affecting the system of oxidative phosphorylation in mitochondria. In the present work, a comparative study of oxygen consumption in 3T3BSV40 cells transformed with oncovirus SV40 and parental BALB/3T3 cells was conducted. Such fractions of oxygen consumption as “phosphorylating” respiration coupled to ATP synthesis, “free” respiration not coupled to ATP synthesis, and “reserve” or hidden respiration observed in the presence of protonophore were determined. Maximal respiration was shown to be only slightly decreased in 3T3BSV40 cells as compared to BALB/3T3. However, in the case of certain fractions of cellular respiration, the changes were significant. “Phosphorylating” respiration was found to be reduced to 54% and “reserve” respiration, on the contrary, increased up to 160% in virustransformed 3T3BSV40 cells. The low rate of “phosphorylating” respiration and high “reserve” respiration indicate that under normal incubation conditions the larger part of mitochondrial respiratory chains of the virustransformed cells is in the resting state (i.e. there is no electron transfer to oxygen). The high “reserve” respiration is suggested to play an important role in preventing apop tosis of 3T3BSV40 cells. DOI: 10.1134/S0006297913080063 Key words: BALB/3T3 and 3T3BSV403T3 cell cultures, respirometry, cellular respiration, inhibitors of oxidative phospho rylation, tumor cells, programmed cell death
We have recently witnessed growing interest in stud ies of bioenergetics of cells with different levels of trans formation due to the possibility of tumor therapy based on influencing the systems of glycolytic and oxidative phosphorylation [15]. Many studies have focused on clarifying the reasons for the relatively high glycolysis and decreased respiration in tumor cells – phenomena first discovered by Warburg [6]. It has been established that the high level of glycolysis is associated with increased expression of a number of genes, this fact leading to the Abbreviations: MPTP, mitochondrial permeability transition pores; MRC, mitochondrial respiratory chains; PCD, pro grammed cell death; RC, respiratory control coefficients; RCADP, control of respiration by the system of ATP synthesis from ADP and phosphate; RCPMF, control of respiration by proton motive force. * To whom correspondence should be addressed.
activation of glucose transport and its utilization in the cells. This ensures efficient energy supply of tumor cells, particularly under hypoxic conditions [4]. As for the decrease in respiration level and its importance for tumor cells, this issue still remains controversial. Relatively low oxygen consumption and reduced activity of certain mitochondrial enzymes have been found in some trans formed cells, but low oxygen consumption has been also shown to be not a compulsory feature of tumor cells [1, 2, 4]. Study of oxygen consumption in mitochondria of tumor cells is of considerable interest due to the fact that mitochondria are responsible for the initial stages of pro grammed cell death (PCD), in particular for the develop ment of apoptosis and the release of apoptosisinducing factors into the cytosol [7, 8]. Suppression of PCD is now regarded as one of the main reasons for the high level of survival of tumor cells [8, 9].
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RESPIRATION OF 3T3 CELLS Some inconsistency in the data on bioenergetics of tumor cells is due to the fact that in a number of studies their respiration was compared to glycolysis only on a sin gle cell model, while in other cases, including the studies by Warburg, it is bioenergetics of tumor and normal cells of different origin that was the object of research. In this work, we compare respiration of homologous BALB/3T3 cells. We used a standard cell line of parental BALB/3T3 fibroblasts and a standard culture of the same cells infect ed with oncogenic SV40 virus as the objects for our study. Different versions of 3T3 cell lines are common materials for a wide range of studies, particularly for studies of virusinduced cell transformation. One of the reasons is that 3T3 cells cultures, depending on their age, have properties of both primary (less than 10 passages) and minimally transformed continuous cultures (passage number higher than 1012). Different 3T3 cell lines were first obtained and studied by Aaronson and Todaro; the same authors showed the possibility of their transforma tion by SV40 virus [10]. Morphokinetics and proliferation of various 3T3 cell lines were also studied and described by Russian researchers [11, 12]. Because oxygen consumption in mitochondria is not only connected to ATP synthesis, but also provides a number of other functions, in this work we studied partial reactions of oxygen consumption by BALB/3T3 and 3T3BSV40 cells. Several respiration fractions have been investigated: “phosphorylating” respiration coupled to ATP synthesis, “free” respiration not coupled to ATP synthesis, and “reserve” or hidden respiration induced by exogenous protonophore. Such a study is relevant as it allows estimating the changes in respiration fractions responsible for different mitochondrial functions in virus transformed and other tumor cells.
MATERIALS AND METHODS Cell cultures. We used mouse embryonic fibroblasts of BALB/3T3 line, clone A31 (hereinafter BALB/3T3) and the same fibroblasts transformed with SV40 virus, 3T3BSV40 cell line. The cultures were certified and obtained from the Russian Collection of Cell Cultures (Institute of Cytology of the Russian Academy of Sciences, St. Petersburg). Conditions for cell cultivation. The cells were cul tured according to standard procedure [13] and passport data for BALB/3T3 and 3T3BSV40. DMEM medium with glutamine and 4.5 g/liter of glucose (MP Biomedical, USA) was used for cell cultivation and incu bation. The following components were added to this medium: 10% fetal bovine serum (HyClone, USA), 1 g/liter sodium bicarbonate, 20 mM Hepes (Sigma, USA), and 80 µg/ml gentamicin (JSC Biokhimik, Russia). Cells were cultured in vials (25 cm2; Greiner, Germany) at 37°C. BALB/3T3 cells were seeded at BIOCHEMISTRY (Moscow) Vol. 78 No. 8 2013
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300,000400,000 cells per vial with 5 ml of medium and were removed after 34 days using trypsinEDTA solution (SPE PanEco). 3T3BSV40 cells were seeded at 400,000 500,000 cells per vial with 5 ml of medium and were removed after 34 days using a solution of 0.25% trypsin and 0.02% Versene (ratio 1 : 3). After removal, the cells were suspended in DMEM medium with the aforemen tioned additives (106 cells per ml of incubation medium). The number of cells was counted in a Goryaev chamber after trypan blue staining. The cells obtained in the labo ratory after 1220 passages were used for the experiments. Oxygen consumption by cells was studied using an Oxygraph2k device (OROBOROS Instruments, Austria) according to the recommended protocol [14, 15]. The Oxygraph2k device was used in standard configuration with two chambers of 2 ml each. Agitator speed was 750 rpm; temperature of the incubation medium was 37°C. The DatLab4 program was used for data collection and analysis. Cell incubation conditions. Cellular respiration was determined in cells incubated in DMEM medium of the abovedescribed composition. Cell concentration in each chamber was ≈106 cells per ml of the incubation medium. Oligomycin (Sigma) was used as an inhibitor of ATP synthesis, and carbonyl cyanide 4(trifluorometh oxy)phenylhydrazone (FCCP; Sigma) was used as a protonophoric uncoupler of oxidative phosphorylation. Oxygen consumption rate was measured in the absence of inhibitors of oxidative phosphorylation and in the pres ence of oligomycin (1 µg/ml of medium) and FCCP (2.5 µM) added to the incubation medium. Concentrations of FCCP and oligomycin were chosen on the basis of titration of respiration by these inhibitors. Maximal activation of respiration (100%) in 3T3BSV40 cells and 9095% activation of respiration in BALB/3T3 cells were observed at the selected FCCP concentration. In the case of selected oligomycin concentration, maxi mal inhibition of respiration was observed in both BALB/3T3 and 3T3BSV40 cells. Statistical analysis of experimental results. The Graph Pad Prisma 4 program was used for statistical analysis of the data. The samples were compared using the nonpara metric Mann–Whitney Utest. The differences were con sidered reliable at p < 0.05 (*). The tables show the mean values ± standard error of the means (the latter were cal culated from the data of 68 experiments for all the param eters of cellular respiration presented in this work). Determination and calculation of partial reactions of cellular respiration. The following partial rates of oxygen consumption by cells were measured and calculated: Vnorm – “normal respiration” or the base rate of oxygen consumption by cells under normal incubation condi tions in the absence of inhibitors and uncouplers of oxidative phosphorylation; Vfree – “free respiration” or the rate of oxygen consumption by cells measured in the presence of oligomycin, the inhibitor of mitochondrial
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FoF1ATP synthase; Vphosph – “phosphorylating respira tion” or oxygen consumption coupled to ATP synthesis carried out by mitochondrial FoF1ATP synthase, calcu lated as the difference between Vnorm and Vfree; Vmax – “maximal respiration” or the maximum possible rate of oxygen consumption by cells, this rate being determined in the presence of FCCP, the protonophore and uncou pler of oxidative phosphorylation; Vres – “reserve respira tion” or the rate of oxygen consumption calculated as the difference between Vmax and Vnorm. Glucose consumption was determined by using the test system AccutrendPlus (Roche Diagnostics). Glucose consumption was shown to be 1.2 and 5.5 nmol/min per 106 cells for BALB/3T3 and 3T3BSV40, respectively, when cells were cultured under aerobic conditions in the absence of inhibitors of oxidative phosphorylation.
RESULTS Partial respiration reactions of minimally transformed BALB/3T3 cells and virustransformed 3T3BSV40 cells. In this work, we comparatively studied the partial reactions of oxygen consumption by BALB/3T3 and 3T3BSV40 cells. We determined the rate of oxygen consumption by the cells under normal conditions in the incubation medi um, in the presence of oligomycin inhibiting ATP synthe sis, and in the presence of a saturating concentration of the protonophore FCCP. The data are shown in Table 1. Baseline oxygen consumption by cells under normal conditions, Vnorm, provides energy for ATP synthesis and other processes in mitochondria that require energy. The Vnorm value was shown to be reliably lowered in 3T3B SV40 cells (≈30%) when compared to minimally trans formed BALB/3T3 cells. “Free” oxygen consumption, i.e. not coupled to ATP synthesis in mitochondrial respi ratory chain (MRC), Vfree, was determined when FoF1 ATP synthase was blocked by oligomycin. The released energy is used for ion transport, including futile ion trans port, which produces heat. Vfree was shown to be slightly raised in 3T3BSV40 cells. “Phosphorylating” respiration provides energy for ATP synthesis in MRC. Vphosph was shown to be reduced almost twofold in 3T3SV40 cells
compared to BALB/3T3 cells. The maximal possible rate of oxygen consumption, Vmax, was determined in the presence of a saturating concentration of the protono phore FCCP. The averaged data for Vmax showed this parameter to be reduced by 17% in 3T3BSV40 cells. The fraction of respiration activated by FCCP, which we call “reserve respiration”, Vres, was also determined. This res piration remains “hidden” or “reserved” under normal conditions of cell incubation in the absence of exogenous protonophore. Vres was found to be reliably increased by almost 1.6fold in 3T3BSV40 cells. Functional analysis of partial reactions of oxygen con sumption in minimally transformed and virustransformed cultures of 3T3 cells. To assess the functional state of mitochondria in the cells, characteristics such as coeffi cients of “respiratory control”, i.e. control of respiration by the system of ATP synthesis from ADP and phosphate (RCADP) and control of respiration by the proton motive force (RCPMF), were calculated. The results are presented in Table 2. Respiratory control coefficient, RCADP, is widely used in bioenergetics in experiments on isolated mito chondria; it is calculated as the ratio of oxygen consump tion rates in the presence and in the absence of ADP or before and after inhibition of ATP synthesis by oligomycin. RCADP characterizes the degree of coupling between respiration and phosphorylation. As presented in Table 2, the traditional way of calculation (RCADP = Vnorm/Vfree) shows a relatively high value of respiratory control in BALB/3T3 cells, RCADP = 5.6. RCADP 4 is considered high in isolated mitochondria of normal cells. In the case of 3T3BSV40 cells, RCADP coefficient is reduced approximately twofold, this indicating a low degree of coupling between respiration and phosphoryla tion, i.e. in this case a significantly smaller portion of oxy gen consumed by cells is used for ATP synthesis. Control of respiration by proton motive force (RCPMF) is another parameter showing significant differ ences in the regulation of energy supply systems in BALB/3T3 and 3T3BSV40 cells. This value was calcu lated as the level of activation of the basic respiration by FCCP. This protonophore increases the leakage of H+ ions and decreases the proton motive force on the inner
Table 1. Partial rates of oxygen consumption by BALB/3T3 and 3T3BSV40 cells under different incubation condi tions (n = 68) Rate of oxygen consumption by cells, pmol/s per 106 cells Cells Vnorm
Vfree
Vphosph
Vmax
Vres
BALB/3T3
45.7 ± 9.7
9.8 ± 2.7
35.9 ± 10.1
58.7 ± 10.5
12.9 ± 3.3
3T3BSV40
30.8 ± 9.4*
11.5 ± 3.2
19.3 ± 7.1**
48.9 ± 8.8
20.4 ± 4.3*
Note: Statistically significant differences between the respiration in 3T3BSV40 and BALB/3T3 cells are indicated: * p < 0.05; ** p < 0.01.
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Table 2. Respiratory control coefficients in BALB/3T3 and 3T3BSV40 cells under different incubation conditions (n = 68) Respiratory control coefficients in cells Cells RCADP (Vnorm/Vfree)
Ratio Vphosph/Vfree
RCPMF (Vmax/Vnorm)
Ratio Vres/Vnorm
BALB/3T3
5.59 ± 1.51
4.59 ± 1.51
1.29 ± 0.06
0.29 ± 0.06
3T3BSV40
2.68 ± 0.39**
1.58 ± 0.43**
1.78 ± 0.23**
0.78 ± 0.23**
Changes in RC coefficients in 3T3BSV40*
48%
34%
138%
273%
* RC values in BALB/3T3 cells were assumed to be 100%. ** Statistically significant differences between the respiration in 3T3BSV40 and BALB/3T3 cells are indicated: p < 0.01.
mitochondrial membrane. The protonophore only slight ly activates respiration in BALB/3T3 cells (RCPMF = 1.3). However, in 3T3BSV40 cells it caused a significantly higher activation of respiration (RCPMF = 1.8). The revealed differences in RCADP and RCPMF values are well supported and are much more significant when individual components of respiration are analyzed, Vphosph instead of Vnorm and Vres instead of Vmax, respectively. As shown in Table 2, in this case the Vphosph/Vfree value is low ered threefold, and Vres/Vnorm value is increased almost threefold in 3T3BSV40 cells. Ratio between “phosphorylating”, “free”, and “reserve” oxygen consumption by BALB/3T3 cells and virustransformed 3T3BSV40 cells. As shown above in Table 1, the maximal rates of oxygen consumption in the presence of protonophore, Vmax, have similar values in BALB/3T3 and 3T3BSV40 cells. These data suggest that the potential ability of mitochondria to transfer electrons along the respiratory chain is not significantly changed in 3T3BSV40 when compared to BALB/3T3 cells. As Vmax is composed of three main components, “phosphorylat ing”, “free”, and “reserve” respiration, contribution of these three components in the maximal possible respira tion was estimated. Normalized values of Vphosph, Vfree, and Vres were calculated in each experiment, and average val ues were calculated based on these data. Normalized Vphosph and Vres values were shown to be strongly altered in 3T3BSV40 cells. In the case of BALB/3T3 cells, Vphosph constitutes 63% of Vmax, while in 3T3BSV40 cells this component is only 36% of Vmax. Due to restriction of Vphosph, mitochondrial respiratory potential in 3T3BSV40 cells is not fully realized under normal conditions. This observation is consistent with the fact that the protonophoreactivated fraction of respiration, Vres, con stitutes about 20% of Vmax in BALB/3T3 cells, while in the case of 3T3BSV40 cells this fraction is substantially increased, up to 41% of Vmax. “Free” respiration, Vfree, changed only slightly; it was shown to be 17 and 23% of Vmax in BALB/3T3 and 3T3BSV40 cells, respectively. BIOCHEMISTRY (Moscow) Vol. 78 No. 8 2013
DISCUSSION Partial reactions of cellular respiration of 3T3BSV40 cells transformed by Simian virus 40 and the parental BALB/3T3 cells have been comparatively studied. SV40 is generally accepted to be an oncogenic virus capable of inducing malignant transformation of different types of human and animal cells, including 3T3 cells [1618]. Tumor transformation is shown to occur due to the fact that SV40 T antigen binds tumor suppressor proteins such as pRb and p53 in host cells, these proteins being the key regulators of cell proliferation involved in arrest of the cell cycle and induction of PCD [1, 18, 19]. 3T3 cells infected by SV40 virus show signs of tumor cells: accelerated divi sion, decreased cell death, lower dependence of cell growth on the presence of serum in the cultivation medium, loss of contact inhibition of growth, increased glucose utilization and lactate production [2023]. In this connection, the approach used in this work to study partial reactions of res piration and the resulting data may be useful for under standing the role of cellular respiration not only in virus transformed cells, but also in other types of tumor cells. We observed a relatively small but significant decrease in basic respiration (Vnorm) in 3T3BSV40 cells. A similar decrease in basic respiration in 3T3 cells infect ed with oncogenic HRAS virus was described by Yang et al. [24]. We found a significant decrease in phosphorylat ing respiration (Vphosph) in 3T3BSV40 compared to BALB/3T3. Since the rate of phosphorylating respiration in 3T3BSV40 cells is less than the half of the maximal possible respiration rate, we can assume that electron transfer along the mitochondrial respiratory chains (MRC) is not the cause and limiting stage of the low Vphosph. There seem to be two possible reasons for the low Vphosph in 3T3BSV40 cells: a wellknown for tumor cells suppression of respiration by increased glycolysis as a result of competition for ADP and Pi (the Crabtree effect [25]) and reduced activity of mitochondrial FoF1ATP synthase caused by decreased expression of its βsubunit
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[26]. This conclusion is supported by the observation that under our experimental conditions glucose consumption was significantly increased in 3T3BSV40 cells. The high reserve respiration (Vres) found in this study is another principal difference of 3T3BSV40 cells. Due to the low rate of Vphosph, a significant part of the MRC does not transfer electrons to oxygen, being in a quiescent or reserve state (when 3T3BSV40 cells are incubated under normal conditions). In the absence of exogenous protonophore, arrest of electron transfer in reserve MRC should lead to an increased reduction level of carriers of electrons and hydrogen, such as NADH, NADPH, QH2, etc. Significantly increased NADH/NAD ratio in 3T3 SV40 cells, 34 times higher than in parental 3T3 cells, was shown by Schwartz et al. [27]. In experiments with isolated mitochondria and mitochondria in situ in cultured cells, MPTP opening and release of apoptosisinducing factors from mitochondria were shown to be coupled to the reduction of membrane potential and NADH oxidation [3, 7, 2831]. Hence, it seems reasonable to suggest that high “reserve respira tion” of 3T3SV40 cells found in this study may prevent MPTP opening, and thus this phenomenon cannot be viewed as a simple physiologically insignificant conse quence of reduced “phosphorylating” respiration; it rather plays a significant role in increased survival of virustransformed cells. It should be noted that the con cept of “reserve respiration” used in this work is analo gous to the concept of “respiratory capacity” (the differ ence between the maximal and base respiration) suggest ed and successfully applied by Nicholls et al. [32, 33] to estimate the survival of nerve cells under conditions of maximal physiological stimulus and in neuropathologies. The approach to the study of partial reactions of oxy gen consumption discussed in this paper seems to be quite useful for studies of bioenergetics of other transformed and tumor cells. Increased “reserve respiration” found in 3T3SV40 cells can be regarded as one of the targets for anticancer drugs. In this connection, it seems interesting to note that Chen et al. [33] reported that a number of nat ural anticancer compounds are currently undergoing clin ical trials, these drugs being aimed at correction of mito chondrial functions, including several compounds capable of decreasing mitochondrial membrane potential. REFERENCES 1. 2. 3. 4.
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