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Effects of Genistein on ATP Induced DNA Synthesis and Intracellular Alkalinization in Chang Liver Cells
Effects and
of Genistein Intracellular
on ATP
Induced
Alkalinization
Kwok-Hung
Sit,
Kim-Ping
DNA
in Chang
Wong'
and
Boon-Huat
Synthesis Liver
Cells
Bay
Department of Anatomy and 'Department of Biochemistry, National University of Singapore, Kent Ridge, Singapore 0511 Received
April
19, 1991
Accepted
May 30, 1991
ABSTRACT Extracellular ATP (adenosine 5'-triphosphate) produces a dose-depend ent elevation of DNA synthesis concomitantly with raised intracellular pH (pH;) in quiescent monolayer Chang liver cells. Genistein produces a dose-dependent suppres sion of ATP-induced DNA synthesis without affecting cell viability (dead cell index 5%) or pH, elevation. The results suggest that (a) mitogenesis and pH; are not im perative correlates and (b) genistein is effective in inhibiting DNA synthesis whether due to growth factor or "non-growth factor" stimulation.
Intracellular alkalinization has been corre lated with cell proliferation (1). At least some of the reactions of the proliferative cascade appear to be mediated directly by Na+/H+ exchanges (2) which in turn seem linked to the transformation-sensitive cyclin-protein kinase complex and mitosis specific phosphorylations via growth factors and cdc2-protein kinase, a major cell cycle regulated tyrosine kinase sub strate (3, 4). Here we show that ATP, which is not a growth factor per se, is capable of stimulating DNA synthesis in quiescent cells. With that, we further show that genistein can completely dissociate DNA synthesis from intracelluar alkalinization. Genistein, a tyrosine specific protein kinase and DNA topoisomerase II in hibitor (5, 6), thus appears to be effective in inhibiting DNA synthesis stimulated by growth promoters, irrespective of whether they are growth factors or not. Monolayer cultures of Chang liver cells (ATCC CCL 13) were grown in Dulbecco's modified Eagle's medium (DMEM, Sigma) with 10% fetal bovine serum (FBS, Sera-lab). Parallel cultures were seeded in 25-cm2 flasks
(Costar) at 3 X 105cells/flask from a stirred stock suspension. For the [3H]thymidine uptake study, parallel confluent quiescent cultures were labelled with 1 ml buffer containing [methyl-3H]thymidine (spec. act. of 72 Ci/mmol, ICN) and incu bated with the appropriate concentration of ATP (Sigma) and genistein (Funakoshi Phar maceutical). The genistein stock solution was 50 mM in DMSO (dimethylsulfoxide, Sigma). The ATP stock solution was 140 mM in H2O, neutralized to pH 7.2 with NaOH. The buffer used was Na+-HEPES (120 mM NaCI and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesul fonic acid JHEPES, Sigma ~). After the pulse incubation, the reaction was stopped by adding an equal volume of 10% TCA (tri chloroacetic acid, Sigma). Cells were scraped and washed with 3 X 10 ml normal saline. Radioactivity in the TCA precipitable material was measured by scintillation counting. For measurement of pH,, the cells were loaded with 10,uM BCECF-am (Molecular Probes) before incubating with 7 mM ATP in Na+-HEPES buffer. The fluorescence change was recorded in the LS-5 Spectrometer (Per
Fig. 1. [3H]Thymidine uptake in ATP dose-response and genistein dose-response. [3H]Thymidine uptake (A) with and without 0.5 mM genistein: ATP dose-response, and (B) in the presence of 10 mM ATP: genis tein dose-response. IC„ = 200,uM. Error bar = 2S.E. (95% confidence limits).
kin Elmer) as previously described (7). Added ATP caused an increase in DNA synthesis in confluent quiescent cultures of Chang liver cells, as shown by the dose-re sponse curve in Fig. IA. ATP at 10 mM pro duced peak level mitogenic stimulation in the culture. At this peak stimulatory level, the genistein dose-response (Fig. 113)gave an IC„ = 200 ,u M (dose which reduced the incorpora tion of [3H]thymidine by 50%). Using 500 uM, we obtained total suppression of DNA synthe sis at all concentrations of applied extracellu lar ATP. The dead cell index averaged around 5% as measured by trypan blue dye inclusion, with or without genistein, indicating that the DNA synthesis suppression was not the result of cell death. Figure 1A also shows that there was a low level of thymidine uptake occuring without addition of exogenous ATP (0 mM). Even this basal activity was suppressed by the added 0.5 mM genistein. This corroborates our earlier observations, namely that confluent parallel cultures of Chang cells maintained in normal DMEM growth medium (with 10% FBS) without exogenous ATP stimulation have their basal [3H]thymidine uptake levels suppressed uniformly by 0.5 mM genistein (8). At this 100% DNA synthesis abolition, however, pH; (as measured by intracellular BCECF fluorescence change) still increased on incubation with 10 mM ATP (Fig. 2). If pH;
Fig. 2. pH; of the culture incubated with 7 mM ATP with and without 0.5 mM genistein. The trace line (ATP and genistein) on the right side is a super imposed insert to show that it is parallel with the left side trace line (ATP) which is without added genistein. BCECF was loaded by prepulse incubation of the es ter.
and
DNA
expected sion
of
synthesis result pH;
were
should
elevation.
correlated, be Such
complete a trend
then
the
suppres did
not
occur.
It has been suggested (9) that genistein in hibits DNA synthesis and PI second messen ger generation caused by growth factors and that genistein does not affect PI second mes senger generation when initiated by "non growth factors" such as ATP and angiotensin II. Here we have shown that genistein is effec tive in suppressing DNA synthesis when stimulated by ATP. The target of genistein in
hibition could be the ATP binding site of tyro sine kinase (5, 10). It has been demonstrated that genistein directly reduces the phosphory lation of oncogenic proteins and other sub strates (11). Alternatively, genistein, being a DNA topoisomerase II inhibitor (6, 10) could directly suppress the replication process. Car cinogenesis is linked to mitogenesis as the rate-limiting factor or at least one of the causative factors (12). Specific suppression of growth factor or "non-growth factor" induced mitogenesis would substantiate the status of genistein as one of the first of a new class of anti-cancer drug (9, 10, 13). However, the occurence of intracellular alkalinization early in the mitogenic process is a consistent, universal finding (2), and there appears to be a linkage between intracellular alkalinization and cell retraction either artifi cally induced (7, 8, 14, 15) or spontaneously as in mitosis. If genistein does not inhibit alka linization concurrently with abolition of DNA synthesis, then it would suggest that cell de tachment, necessary in metastasis (16), may be unaffected.
6
7
8
9
10
11
Acknowledgments We and grant
thank the
Mrs.
National
M.
Singh
University
for of
secretarial Singapore
assistance for
12
research
RP880312.
REFERENCES
13 1 Madshus, I.H.: Regulation of pH in eukaryotic cells. Biochem. J. 250, 1-8 (1988) 2 Grinstein, S., Rotin, D. and Mason, M.J.: Na+/H+ exchange and growth factor-induced cytosolic pH changes. Role in cellular prolifera tion. Biochim. Biophys. Acta 988, 73-97 (1989) 3 Gould, K.L. and Nurse, P.: Tyrosine phosphoryla tion of the fission yeast cdc2 protein kinase reg ulates entry into mitosis. Nature 342, 39-45 (1989) 4 Draetta, G., Piwinica-Worms, H., Morrison, D., Druker, B., Roberts, T. and Bleach, D.: Human cdc2 protein kinase is a major cell cycle regulated tyrosine kinase substrate. Nature 336, 738-744 (1988) 5 Akiyama, R., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M. and Fukami, Y.: Genistein, a specific inhibitor of tyro
14
15
16
sine protein kinase. J. Biol. Chem. 262, 5592 5595 (1987) Okura, A., Arakawa, H., Oka, H., Yoshinari, T. and Monden, Y.: Effect of genistein on topoisomerase activity and on the growth of [val 12] Ha-ras-transformed NIH 3T3 cells. Biochem. Biophys. Res. Commun. 157, 183-189 (1988) Sit, K.H. and Wong, K.P.: Induced interphase cell retraction: its reversal and EGF potentiation. Tis sue Cell 21, 321 333 (1989) Sit, K.H. and Wong, K.P.: Sulphate induces very fast cell rounding and detachment. Biochim. Bio phys. Acta 1092, 180 183 (1991) Dean, N.M., Kanemitsu, M. and Boynton, A.L.: Effects of the tyrosine kinase inhibitor genistein on DNA synthesis and phospholipid-derived second messenger generation in mouse TI/2 fibroblasts and rat liver T51B cells. Biochem. Biophys. Res. Commun. 165, 795-801 (1989) Markovits, J., Linassier, C., Fosse, P., Coup rie, J., Pierre, J., Jacquemin-Sablon, A., Saucier, J.-M., Le Pecq, J.-B. and Larsen, A.K.: Inhibi tory effects of the tyrosine kinase inhibitor gene stein on mammalian topoisomerase II. Cancer Res. 49, 5111 5117 (1989) Nishimura, J., Takahira, H., Shibuta, K., Muta, K., Yamamoto, M., Ideguchi, H., Umemura, T. and Nawata, H.: Regulation of biosynthesis and phosphorylation of P210bcr/ab' protein during dif ferentiation of K562 cells. Leuk. Res. 12, 875 885 (1990) Prelich, G., Kostura, M., Marshak, D.R., Mathews, M.B. and Stillman, B.: The cell cycle regulated proliferating cell nuclear antigen is re quired for SV40 DNA replication in vitro. Nature 326, 471-475 (1987) Linassier, C., Pierre, M., le Pecq, J.B. and Pierre, J.: Mechanisms of action in NIH 3T3 cells of genistein an inhibitor of EGF receptor tyrosine kinase activity. Biochem. Pharmacol. 39, 187-193 (1990) Sit, K.H., Bay, B.H. and Wong, K.P.: Antiport mediated cell retraction: viable rounding and dis tinctive endocytosis. Tissue Cell 22, 785-802 (1990) Sit, K.H., Bay, B.H. and Wong, K.P.: Antiport mediated rounding and endocytosis are enhanced by sulphate: downgradient influx of huge macro molecules sorted into granular patches. Tissue Cell (1991) (in press) Juliano, R.L.: Membrane receptors for extracellu lar macromolecules: Relationship to cell adhesions and tumor metastasis. Biochim. Biophys. Acta 907, 261-278 (1987)
of Genistein Intracellular
on ATP
Induced
Alkalinization
Kwok-Hung
Sit,
Kim-Ping
DNA
in Chang
Wong'
and
Boon-Huat
Synthesis Liver
Cells
Bay
Department of Anatomy and 'Department of Biochemistry, National University of Singapore, Kent Ridge, Singapore 0511 Received
April
19, 1991
Accepted
May 30, 1991
ABSTRACT Extracellular ATP (adenosine 5'-triphosphate) produces a dose-depend ent elevation of DNA synthesis concomitantly with raised intracellular pH (pH;) in quiescent monolayer Chang liver cells. Genistein produces a dose-dependent suppres sion of ATP-induced DNA synthesis without affecting cell viability (dead cell index 5%) or pH, elevation. The results suggest that (a) mitogenesis and pH; are not im perative correlates and (b) genistein is effective in inhibiting DNA synthesis whether due to growth factor or "non-growth factor" stimulation.
Intracellular alkalinization has been corre lated with cell proliferation (1). At least some of the reactions of the proliferative cascade appear to be mediated directly by Na+/H+ exchanges (2) which in turn seem linked to the transformation-sensitive cyclin-protein kinase complex and mitosis specific phosphorylations via growth factors and cdc2-protein kinase, a major cell cycle regulated tyrosine kinase sub strate (3, 4). Here we show that ATP, which is not a growth factor per se, is capable of stimulating DNA synthesis in quiescent cells. With that, we further show that genistein can completely dissociate DNA synthesis from intracelluar alkalinization. Genistein, a tyrosine specific protein kinase and DNA topoisomerase II in hibitor (5, 6), thus appears to be effective in inhibiting DNA synthesis stimulated by growth promoters, irrespective of whether they are growth factors or not. Monolayer cultures of Chang liver cells (ATCC CCL 13) were grown in Dulbecco's modified Eagle's medium (DMEM, Sigma) with 10% fetal bovine serum (FBS, Sera-lab). Parallel cultures were seeded in 25-cm2 flasks
(Costar) at 3 X 105cells/flask from a stirred stock suspension. For the [3H]thymidine uptake study, parallel confluent quiescent cultures were labelled with 1 ml buffer containing [methyl-3H]thymidine (spec. act. of 72 Ci/mmol, ICN) and incu bated with the appropriate concentration of ATP (Sigma) and genistein (Funakoshi Phar maceutical). The genistein stock solution was 50 mM in DMSO (dimethylsulfoxide, Sigma). The ATP stock solution was 140 mM in H2O, neutralized to pH 7.2 with NaOH. The buffer used was Na+-HEPES (120 mM NaCI and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesul fonic acid JHEPES, Sigma ~). After the pulse incubation, the reaction was stopped by adding an equal volume of 10% TCA (tri chloroacetic acid, Sigma). Cells were scraped and washed with 3 X 10 ml normal saline. Radioactivity in the TCA precipitable material was measured by scintillation counting. For measurement of pH,, the cells were loaded with 10,uM BCECF-am (Molecular Probes) before incubating with 7 mM ATP in Na+-HEPES buffer. The fluorescence change was recorded in the LS-5 Spectrometer (Per
Fig. 1. [3H]Thymidine uptake in ATP dose-response and genistein dose-response. [3H]Thymidine uptake (A) with and without 0.5 mM genistein: ATP dose-response, and (B) in the presence of 10 mM ATP: genis tein dose-response. IC„ = 200,uM. Error bar = 2S.E. (95% confidence limits).
kin Elmer) as previously described (7). Added ATP caused an increase in DNA synthesis in confluent quiescent cultures of Chang liver cells, as shown by the dose-re sponse curve in Fig. IA. ATP at 10 mM pro duced peak level mitogenic stimulation in the culture. At this peak stimulatory level, the genistein dose-response (Fig. 113)gave an IC„ = 200 ,u M (dose which reduced the incorpora tion of [3H]thymidine by 50%). Using 500 uM, we obtained total suppression of DNA synthe sis at all concentrations of applied extracellu lar ATP. The dead cell index averaged around 5% as measured by trypan blue dye inclusion, with or without genistein, indicating that the DNA synthesis suppression was not the result of cell death. Figure 1A also shows that there was a low level of thymidine uptake occuring without addition of exogenous ATP (0 mM). Even this basal activity was suppressed by the added 0.5 mM genistein. This corroborates our earlier observations, namely that confluent parallel cultures of Chang cells maintained in normal DMEM growth medium (with 10% FBS) without exogenous ATP stimulation have their basal [3H]thymidine uptake levels suppressed uniformly by 0.5 mM genistein (8). At this 100% DNA synthesis abolition, however, pH; (as measured by intracellular BCECF fluorescence change) still increased on incubation with 10 mM ATP (Fig. 2). If pH;
Fig. 2. pH; of the culture incubated with 7 mM ATP with and without 0.5 mM genistein. The trace line (ATP and genistein) on the right side is a super imposed insert to show that it is parallel with the left side trace line (ATP) which is without added genistein. BCECF was loaded by prepulse incubation of the es ter.
and
DNA
expected sion
of
synthesis result pH;
were
should
elevation.
correlated, be Such
complete a trend
then
the
suppres did
not
occur.
It has been suggested (9) that genistein in hibits DNA synthesis and PI second messen ger generation caused by growth factors and that genistein does not affect PI second mes senger generation when initiated by "non growth factors" such as ATP and angiotensin II. Here we have shown that genistein is effec tive in suppressing DNA synthesis when stimulated by ATP. The target of genistein in
hibition could be the ATP binding site of tyro sine kinase (5, 10). It has been demonstrated that genistein directly reduces the phosphory lation of oncogenic proteins and other sub strates (11). Alternatively, genistein, being a DNA topoisomerase II inhibitor (6, 10) could directly suppress the replication process. Car cinogenesis is linked to mitogenesis as the rate-limiting factor or at least one of the causative factors (12). Specific suppression of growth factor or "non-growth factor" induced mitogenesis would substantiate the status of genistein as one of the first of a new class of anti-cancer drug (9, 10, 13). However, the occurence of intracellular alkalinization early in the mitogenic process is a consistent, universal finding (2), and there appears to be a linkage between intracellular alkalinization and cell retraction either artifi cally induced (7, 8, 14, 15) or spontaneously as in mitosis. If genistein does not inhibit alka linization concurrently with abolition of DNA synthesis, then it would suggest that cell de tachment, necessary in metastasis (16), may be unaffected.
6
7
8
9
10
11
Acknowledgments We and grant
thank the
Mrs.
National
M.
Singh
University
for of
secretarial Singapore
assistance for
12
research
RP880312.
REFERENCES
13 1 Madshus, I.H.: Regulation of pH in eukaryotic cells. Biochem. J. 250, 1-8 (1988) 2 Grinstein, S., Rotin, D. and Mason, M.J.: Na+/H+ exchange and growth factor-induced cytosolic pH changes. Role in cellular prolifera tion. Biochim. Biophys. Acta 988, 73-97 (1989) 3 Gould, K.L. and Nurse, P.: Tyrosine phosphoryla tion of the fission yeast cdc2 protein kinase reg ulates entry into mitosis. Nature 342, 39-45 (1989) 4 Draetta, G., Piwinica-Worms, H., Morrison, D., Druker, B., Roberts, T. and Bleach, D.: Human cdc2 protein kinase is a major cell cycle regulated tyrosine kinase substrate. Nature 336, 738-744 (1988) 5 Akiyama, R., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M. and Fukami, Y.: Genistein, a specific inhibitor of tyro
14
15
16
sine protein kinase. J. Biol. Chem. 262, 5592 5595 (1987) Okura, A., Arakawa, H., Oka, H., Yoshinari, T. and Monden, Y.: Effect of genistein on topoisomerase activity and on the growth of [val 12] Ha-ras-transformed NIH 3T3 cells. Biochem. Biophys. Res. Commun. 157, 183-189 (1988) Sit, K.H. and Wong, K.P.: Induced interphase cell retraction: its reversal and EGF potentiation. Tis sue Cell 21, 321 333 (1989) Sit, K.H. and Wong, K.P.: Sulphate induces very fast cell rounding and detachment. Biochim. Bio phys. Acta 1092, 180 183 (1991) Dean, N.M., Kanemitsu, M. and Boynton, A.L.: Effects of the tyrosine kinase inhibitor genistein on DNA synthesis and phospholipid-derived second messenger generation in mouse TI/2 fibroblasts and rat liver T51B cells. Biochem. Biophys. Res. Commun. 165, 795-801 (1989) Markovits, J., Linassier, C., Fosse, P., Coup rie, J., Pierre, J., Jacquemin-Sablon, A., Saucier, J.-M., Le Pecq, J.-B. and Larsen, A.K.: Inhibi tory effects of the tyrosine kinase inhibitor gene stein on mammalian topoisomerase II. Cancer Res. 49, 5111 5117 (1989) Nishimura, J., Takahira, H., Shibuta, K., Muta, K., Yamamoto, M., Ideguchi, H., Umemura, T. and Nawata, H.: Regulation of biosynthesis and phosphorylation of P210bcr/ab' protein during dif ferentiation of K562 cells. Leuk. Res. 12, 875 885 (1990) Prelich, G., Kostura, M., Marshak, D.R., Mathews, M.B. and Stillman, B.: The cell cycle regulated proliferating cell nuclear antigen is re quired for SV40 DNA replication in vitro. Nature 326, 471-475 (1987) Linassier, C., Pierre, M., le Pecq, J.B. and Pierre, J.: Mechanisms of action in NIH 3T3 cells of genistein an inhibitor of EGF receptor tyrosine kinase activity. Biochem. Pharmacol. 39, 187-193 (1990) Sit, K.H., Bay, B.H. and Wong, K.P.: Antiport mediated cell retraction: viable rounding and dis tinctive endocytosis. Tissue Cell 22, 785-802 (1990) Sit, K.H., Bay, B.H. and Wong, K.P.: Antiport mediated rounding and endocytosis are enhanced by sulphate: downgradient influx of huge macro molecules sorted into granular patches. Tissue Cell (1991) (in press) Juliano, R.L.: Membrane receptors for extracellu lar macromolecules: Relationship to cell adhesions and tumor metastasis. Biochim. Biophys. Acta 907, 261-278 (1987)