Selective Effects by Valinomycin on Cytotoxicity ... - Cancer Research

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GROWTH INHIBITION AND CYTOTOXICITY BY VAL 10 15 Chart 9. Effect of daily application of 20 MMVAL in fresh medium containing 5% fetal calf serum on density (top) of ts RSV-Rat-1 cells at 35Â°Cand their relative fraction G,w with G, equivalent of DNA (bottom). Abscissa, time of growth in days (f/d). O, untreated control; A, VAL-treated cells. Table 1 Effect of 24-h treatment with VAL on cellular ATP contents Cells3T3SV40-3T33T6TreatmentControl20 VALControl20 nw VALControl20 nw content(fmol/cell)5.66 (12f3.84 + 0.82" (7)3.05 Â±0.52 (8)2.23 Â±0.27 (4)3.72 Â±0.30 nmiVALATP (7)2.11 Â±0.34 Â±0.20 (4)% * Average Â±SD. 6 Numbers in parentheses, number of determinations. of de creaserelative tocontrol322743 <strong>by</strong> about one-third relative to control if 20 nMVAL is applied for 24 h. Cellular ATP contents 48 h after application of 20 nM VAL (data not shown) within experimental error do not differ from those at 24 h of exposure. DISCUSSION Our present work has shown that daily application of VAL at >2 nM leads to inhibition of proliferation of all cell lines tested. This inhibitory effect saturates at about 20 to 100 nw VAL. The action of VAL is not cytotoxic to nontransformed cells, such as 3T3 and Rat-1, but is cytotoxic to transformed cells with increas ing impact in the order 3T6, PY-3T3, ts RSV-Rat-1 (35Â°C),and SV40-3T3 cells. The low cytotoxic effect on 3T6 and PY-3T3 in contrast to SV40-3T3 cells correlates with their fairly short time lag of 5 to 20 days for induction of tumors upon inoculation into nude mice as compared to >80 days for SV40-3T3 cells (26).5 Possibly, the long latency for tumor development of SV40-3T3 inocula in nude mice results from the death of a majority of the inoculated SV40-3T3 cell population before tumor development starts from a surviving minority of cells. 5 Unpublished data. If SV40-3T3 cells are grown in vitro to saturating cell densities, massive cell death is observed after a delay of 1 or 2 days (1, 2, 4). The growth behavior of 3T6 cells is quite different, because their saturation is reached more gradually, is regulated <strong>by</strong> the serum concentration, and does not exhibit substantial cell death. Similarly, proliferation of 3T6 is inhibited <strong>by</strong> different concentra tions of VAL at graduated cell densities and without substantial cell death (Chart 7). Thus, comparing the growth behavior of different cell lines with and without VAL, the essential action of VAL appears to be to impose on the cells at low growth densities a state of limiting growth condition which normally is encountered only at high cell densities and/or low serum concentration. This interpretation can be formulated quantitatively <strong>by</strong> using a simple population-dynamic model allowing for only 2 cellular subpopu lations, dividing and long-term growth-inhibited (quiescent) cells with a cell density-dependent probability of transition from the dividing to the quiescent subpopulation (27, 28). We have shown earlier that proliferation of 3T3 and SV40-3T3 cells under the usual in vitro conditions may thus be described adequately and that the specific features of SV40-3T3 cells may be accounted for <strong>by</strong> a somewhat lower transition probability (about one-fifth as compared to normal 3T3) to the growth-inhibited state and, above all, <strong>by</strong> a larger rate constant (0.5 to 3.0/day) of death of the growth-inhibited cell population (27, 28). If the major effect of saturating concentrations of VAL is a massive increase of the probability of transition to the growth-inhibited state, cells will accumulate in this state. Then, the rate constant of overall cell death is given <strong>by</strong> that of the inhibited cell subpopulation which, according to Chart 2, is at least 0.6/day in reasonable agreement with the figures for untreated SV40-3T3 cells given above. Even more detailed features, such as the interplay of cell density and action of VAL at different concentrations (cf. Chart 7), may be specified quantitatively according to this model.6 Our data on the effect of VAL on the cellular distribution over the phases of the cell cycle generally agree with this interpreta tion but allow for a more detailed characterization of the mode of growth arrest. Most significantly, all the nontransformed cells are growth inhibited <strong>by</strong> VAL in GÃ¬, whereas all transformed cells are inhibited predominantly in S and G2,although some of them (3T6 and, to a lesser extent, PY-3T3) without VAL are growth arrested in GÃ¬. This apparently general difference between nor mal and transformed cells of the cell cycle phase of growth arrest <strong>by</strong> VAL appears to be relevant for a possible exploitation in phase-specific selective killingof tumor cells in antitumor therapy. The cycle phase of growth arrest <strong>by</strong> VAL generally does not correlate with its cytotoxic effect; e.g., 3T6 cells are arrested in S and G2 <strong>by</strong> VAL but experience only relatively little cytotoxic effect, if compared with SV40-3T3 or ts RSV-Rat-1 (35Â°C)cells. According to our still incomplete data, a better correlation is observed between viability in the presence of VAL and the ability of cells to down-regulate their rRNA metabolism. We have observed5 that 3T3, Rat-1, and 3T6 cells under the action of VAL exhibit a decrease of cellular rRNA similar to that of the untreated controls (23). For 3T6 cells, this result is quite unex pected, inasmuch as they down-regulate their rRNA metabolism but are growth arrested mostly outside the GÃ¬phase of the cell cycle (Chart 7). In contrast, both SV40-3T3 (23) and ts RSV-Rat- 1 (35Â°C)cells5 are not able to regulate their rRNA content â€¢ To be published elsewhere. CANCER RESEARCH VOL. 45 JULY 1985 3026 Downloaded from cancerres.aacrjournals.org on December 2, 2012 Copyright © 1985 American Association for Cancer Research
dependent on cell density (without application of VAL) and thus exhibit features of "unbalanced growth" (29, 30). We have not yet obtained data on the rRNA content of these cells under the action of VAL but would expect that they also exhibit unbalanced growth under these conditions. Multiple studies have shown that fairly high concentrations (>1 ÃÂ¿M) of VAL are required to affect 1C transport through the plasma membrane (31, 32). Mitochondrial function is much more sensitive to VAL; here, concentrations of about 7 to 70 nw have been found to affect the mitochondrial membrane potential (13, 33). In order to arrive at meaningful comparisons between effec tive VAL concentrations of different studies, the strongly lipophilic nature of this compound must be taken into account. The partition coefficient of VAL between phospholipid and water has been determined in bilayer studies and was found to be in the range of 10" to 10s (34). Using the data given in Ref. 13, we have a nearly saturating effect of VAL at 10 Â¿Â¿g VAL/g mitochon drial protein suspended at 7 mg protein/ml, yielding an overall concentration of VAL of about 70 nw. Taking into account 200 tig lipid/mg protein for these mitochondria (35) and a lipid density of about 0.5 g/ml, we arrive at an effective concentration of about 25 UM VAL in mitochondrial nearly saturating effect if a partition lipid under conditions of a coefficient of 104 to 105 is used. If VAL is applied to cell cultures, it is partitioned between water and all lipid phases present, such as serum lipids and cellular lipids. Using cellular lipid contents determined earlier (36), a serum concentration of 5% with total lipid at 2.5 mg/ml serum,5 a lipid density of 0.5 g/ml, and a partition coefficient of 104 to 105 for VAL in all lipids, we arrive at concentrations of VAL in (mitochondrial) lipid of 8 to 80 MMat overall VAL concentrations of 2 to 20 nw, respectively. Thus, the effective VAL concentra tions in (mitochondrial) lipid under the conditions of our present measurements are identical <strong>by</strong> order of magnitude with those found to be effective in reducing mitochondrial membrane poten tial (13), whereas much higher concentrations were needed to affect K+ transport through the plasma membrane (31, 32). Further arguments for a mitochondrial site of action of VAL stem from experiments6 on analogous proliferation-inhibiting effects of mitochondrial uncouplers, such as m-chlorocarbonyl-cyanide phenylhydrazone, at about 10 fiM and on the fast and substantial inhibition of mitochondrial uptake of the cationic fluorescent dye rhodamine 123 upon cellular treatment with 20 nw VAL. Our data given in Table 1 are also consistent with this interpretation, because the ATP content of all cells tested decreases <strong>by</strong> about 30% upon treatment with 20 nw VAL. In order to account for the selective toxic effect of VAL on virus-transformed cells (in the presence of nonselective effects on ATP content), the following possibilities may be discussed. If the action of VAL is mediated via a decrease of cellular ATP content, different cell lines may react differentially to an about equal decrease of cellular ATP. In this context, it is of interest that in both reticulocytes and ascites tumor cells a decline of cellular ATP level <strong>by</strong> 30% leads to inhibition of protein synthesis (37). Alternatively, VAL may act differently on the mitochondrial contribution to intracellular Ca2+ homeostasis (8, 16). Cellular ATP contents may be adjusted <strong>by</strong> concomitant stimulation of glycolysis (38, 39). We have started work to test this second possibility <strong>by</strong> characterizing the effect of VAL on intracellular calcium compartments. By analysis of 45Ca efflux, 3 kinetic calcium compartments may be separated (40), whereas the GROWTH INHIBITION AND CYTOTOXICITY BY VAL intracellular free Ca2+ concentration may be determined using the fluorescent indicator Quin 2 (41). For 3T3 cells, all of these calcium pools were shown to decrease significantly <strong>by</strong> treatment with 20 nw VAL;7 the transformed cell lines are investigated presently. In addition, the effect of VAL on the following cellular reactions appears to represent fruitful issues for further study: interplay between glycolysis and oxidative phosphorylation (37, 38); protein synthesis (37, 42); polyamine metabolism (43); and rRNA metabolism (Ref. 14; own unpublished results briefly dis cussed above). Studies on the exploitation of the cycle phasespecific arrest of tumor cell proliferation also appear to be useful and are in progress in our laboratory using in vitro and in vivo systems. ACKNOWLEDGMENTS We wish to thank Dr. F. Gruber for collaboration and advice in animal experi ments, Dr. G. Stark for discussion, and F. Braun and A. Kesper for their excellent technical assistance. REFERENCES CANCER RESEARCH VOL. 45 JULY 1985 1. Bartholomew, J. C., Yokota, H., and Ross, P. Effect of serum on the growth of Balb 3T3 A31 mouse fibroblasts and an SV40-transformed derivative. J. Cell. Physid., 88: 277-286, 1976. 2. Pardee, A. B. A restriction point for control of normal animal cell proliferation. Proc. Nati. Acad. Sci. USA, 71:1286-1290, 1974. 3. Prescott, D. M. Regulation of cell reproduction. Cancer Res., 28:1815-1820, 1968. 4. Schiaffonati, L, and Baserga, R. Different survival of normal and transformed cells exposed to nutritional conditions nonpermissive for growth. Cancer Res., 37:541-545,1977. 5. Pardee, A. B. Molecular mechanisms of the control of cell growth in cancer. In: C. Nicolini (ed.), Cell Growth, pp. 673-714. New York: Plenum Publishing Corp., 1982. 6. Paul, D., Henahan, M., and Walter, S. Changes in growth control and growth requirements associated with neoplasie transformation in vitro J. Nati. Cancer lnst.,53: 1499-1503,1974. 7. Pedersen, P. L. Tumor mitochondria and the bioenergetics of cancer cells. Prog. Exp. Tumor Res., 22: 190-274, 1978. 8. Bygrave, F. K. Mitochondria and the control of intracellular calcium. Bid. Rev., 53:43-79,1978. 9. Bernal. S. D., Lampidis, T. J., Mclsaac, R. M., and Chen, L. B. Anticarcinoma activity in vivo of rhodamine 123, a mitochondrial-specific dye. Science (Wash. DC), 222:169-172,1983. 10. Lampidis, T. J., Bernal, S. D., Summerhayes, I. C., and Chen, L. G. <strong>Selective</strong> toxicity of rhodamine 123 in carcinoma cells in vitro. Cancer Res., 43: 716- 719,1983. 11. Summerhayes, I. C., Lampidis, T. J., Bernal, S. D., Nadakavukaren, J. J., Nadakavukaren, K. K., Shepherd, E. L., and Chen, L. B. Unusual retention of rhodamine 123 <strong>by</strong> mitochondria in muscle and carcinoma cells. Proc. Nati. Acad. Sci. USA, 79: 5292-5296, 1982. 12. Conover, T. E., and Azzone, G. F. The generation and role of the electric field in (iH* formation and ATP synthesis. In: C. P. Lee, G. Schatz, and G. Dallner (eds.), Mitochondria and Microsomes, pp. 481-528. Reading, MA: Addison Wesley Publishing Co., 1981. 13. Mitchell, P., and Moyte, J. Estimation of membrane potential and pH difference across the cristae membrane of rat liver mitochondria. Eur. J. Biochem., 7: 471-484, 1969. 14. Panchenko, L. F., Stelletskaya, N. V., Syrota, T. V., Bokhon'Ko, A. I., and Schuppe, N. G. <strong>Effects</strong> of inhibitors of energetic metabolism on RNA turnover in animal cells. Biochim. Biophys. Acta, 299: 103-113,1973. 15. Pfaff, E., and Klingenberg, M. Adenine nucleotide translocation of mitochondria. 1. Specificity and control. Eur. J. Biochem., 6: 66-79,1968. 16. Nichoiis. D. G. The regulation of extramitochondrial free calcium ion concentra tion <strong>by</strong> rat liver mitochondria. Biochem. J., 176: 463-474,1978. 17. Azzone, G. F., Pozzan, T., and Massari, S. Proton electrochemical gradient and phosphate potential in mitochondria. Biochim. Biophys. Acta, 570: 307- 315,1978. 18. Smith, G. L. Increased ouabain-sensitive "rubidium* uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplicationstimulating activity. J. Cell Btol., 73: 761-767,1977. 19. Douros, J., and Suffness, M. New natural products under development at the 7T. Hartmann and G. Adam, unpublished data. 3027 Downloaded from cancerres.aacrjournals.org on December 2, 2012 Copyright © 1985 American Association for Cancer Research
Page 1 and 2: Selective
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Page 5: Chart 6. Effect of daily applicatio

Selective Effects by Valinomycin on Cytotoxicity ... - Cancer Research

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