Biochimica et Biophysica Acta, 1132 (1992) 259-264 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
259
BBAEXP 92418
Regulation of topoisomerase II by murine mastocytoma cells Andrew G. Collett and Ray K. Ralph Department of Cellular and Molecular Biology, Unit:ersityof Auckland, Auckland (New Zealand) (Received 24 April 1992)
Key words: Topoisomerase II; Growth; Cytoplasmic factor; (Mouse)
Nuclei from K21 murine mastocytoma cells do not form topoisomerase II-DNA adducts in response to amsacrine in the absence of a cytoplasmic factor tentatively identified as a type of casein kinase (Darkin, S.J. and Ralph, R.K. (1991) Biochim. Biophys. Acta 1088, 285-291). The stimulatory activity was present in extracts from cells grown in horse serum but not in calf serum. Activity was lost following growth arrest by serum deprivation. In contrast, topoisomerase II activity in isolated nuclei did not decline during growth arrest. These results suggest that the resistance of some non-cycling tumour cells to anti-cancer drugs may result from decreased activation of topoisomerase II.
Introduction
D N A topoisomerase II is the nuclear target for a variety of DNA-intercalating anticancer drugs, including the 9-anilino acridine derivative amsacrine [1]. Amsacrine prevents the religation of D N A during the DNA cleavage-resealing reaction catalysed by topoisomerase II and the resulting covalently-linked complexes between DNA and topoisomerase II can be precipitated with K+-sodium dodecylsulphate (SDS) and quantified to measure the activity of the enzyme [2]. Using this technique we demonstrated that isolated nuclei from K21 murine mastocytoma cells would not form topoisomerase II-DNA adducts in response to amsacrine and that a cytoplasmic factor in the ceils facilitated the formation of amsacrine-induced topoisomerase II-DNA complexes (PDC) when cytoplasmic extracts were added to isolated nuclei in vitro [3]. Evidence was also presented that the cytoplasmic factor involved was a 70 kDa protein kinase with properties reminiscent of casein kinase II [4]. Other data also suggest that phosphorylation [5-8], ADP-ribosylation [9], or unidentified factors [10-12] may regulate the activity of topoisomerase II. While investigating the universality and properties of the cytoplasmic factor that activates topoisomerase II in isolated nuclei from mastocytoma cells, we found
Correspondence to: R.K. Ralph, Department of Cellular and Molecular Biology, University of Auckland, Private Bag 92019, Auckland, New Zealand. Abbreviations: SDS, sodium dodecylsulphate; PDC, protein-DNA complexes.
that cytoplasmic extracts from the parent P815 clone of mastocytoma cells did not increase the formation of DNA-topoisomerase II complexes in isolated nuclei treated with amsacrine. Here we show that the different effects of cytoplasmic extracts from K21 and P815 cells are a result of the different serum used to culture the two clones of cells, and that no stimulatory activity is detectable when cytoplasmic extracts are prepared from K21 or P815 ceils grown with neonatal calf serum. Effects of growth arrest by serum deprivation on topoisomerase II in nuclei, and the topoisomerase 1I stimulatory activity in cytoplasmic extracts from K21 mastocytoma ceils grown in horse serum are also described. The results suggest that factors in horse serum induce or activate the cytoplasmic factor (kinase) in mastocytoma cells that is essential for topoisomerase II to respond to amsacrine. Materials and Methods
General methods Mastocytoma cells were grown in suspension in RPMI1640 medium with either neonatal calf serum or horse serum [4], both products of Gibco-BRL (NZ). Cell density was determined with a Neubauer haemocytometer and cell viability by trypan blue exclusion. Proteinase inhibitors used to prepare cytoplasmic extracts were obtained from Boehringer-Mannheim or Sigma and solutions prepared immediately before use. Protein concentrations were measured using Bradford's reagent [13]. During nuclei isolation cell lysis was confirmed by staining nuclei with aniline blue (0.1%, w / v ) dissolved in nuclei isolation buffer (see below).
260 To radiolabel DNA log phase cells (2. l05 cells/ml) were grown with 0.5/~Ci/ml [methyl-3H]thymidine (20 C i / m m o l ) and 1 ~ M thymidine for 16 h. The cells contained approx. 30000 c p m / 1 " 105 cells after precipitation with trichloroacetic acid, collection of the precipitates on G F / C glass fiber filters and measurement in a liquid scintillation spectrometer.
Cytoplasmic extracts Cytoplasmic extracts were prepared from 5 • 108 cells using a slight modification of the procedure of BlankLiss and Schindler [14]. The cells were recovered by centrifugation at 900 x g for 10 min at 2°C, washed twice with ice-cold Tris-buffered saline and the final pellet was resuspended in 17 mM Mops (pH 7), 250 mM sucrose, 2.5 mM EDTA, 400 K I U / m l aprotinin, 0.1 m g / m l leupeptin, 0.01 m g / m l a-macroglobulin, 1 mM phenylmethylsulphonyl fluoride~ 0.1 m g / m l diisopropylfluorophosphate, 2 m g / m l digitonin at 20°C and a final density of 1.2.108 cells/ml. After 10 min at 20°C the lysate was centrifuged at 30000 x g for 10 min at 0°C, the supernatant was recovered, diluted to 4 m g / m l protein and stored as 0.1 ml aliquots at -70°C. Nuclei isolation Nuclei were prepared from 1 - 108 cells pre-labeled with [3H]thymidine. The cells were collected by centrifugation at 1600 x g for 5 min and resuspended in 2 ml of 20 mM Tris-HCl (pH 7.2), 150 mM KC1, 5 mM MgCI 2, 10 mM NazS205, 2% (w/v), Dextran 150-200 then mixed with 2 ml of the same buffer containing (I.1% Triton X-100 in a tight Dounce homogenizer. After four u p / d o w n strokes of the pestle the mixture was kept on ice for 10 rain with occasional gentle vortex mixing. The nuclei were collected by centrifugation at 1 2 0 0 x g for 10 min at 2°C, resuspended to 6" 10 7 nuclei/ml in the nuclei isolation buffer and used immediately. Topoisomerase H assays To measure topoisomerase II activity, intact cells labeled with [3H]thymidine were recovered by centrifugation, washed twice at 37°C with medium containing the same serum used to grow the cells, resuspended in the same medium at 2.105 cells/ml and 1 ml aliquots were distributed into 1.5 ml Eppendorf microfuge tubes at 37°C. The cells were first incubated for 30 min at 37°C then with or without 10 /~M amsacrine for a further 10 min. After centrifugation the cells were resuspended in 1 mi 137 mM NaC1 at 37°C using a vortex mixer and lysed by adding 0.1 ml 12.5% (w/v) SDS, 50 mM E D T A (Na+), 4 m g / m l salmon sperm DNA prewarmed to 65°C. This mixture was gently sucked up and down three times in a siliconized pasteur pipette then incubated at 65°C for 3 min before adding 0.25 ml of 325 mM KCI at 37°C to each tube
and immediately mixing the solutions for l0 s using a vortex mixer at its highest setting. After chilling the tubes on ice for 40 rain the precipitates that formed were centrifuged at 15000 × g for 10 min at 4°C, the pellets were resuspended in 1 ml 10 mM Tris-HC1 (pH 7.5), 100 mM KCI, 5 mM EDTA, 2 p~g/ml salmon sperm DNA, 1 p.M thymidine at 4°C, dispersed using a siliconized pasteur pipette, and collected under gravity on G F / C glass fiber filters presoaked in 5 ~ g / m l salmon sperm DNA. After rinsing the tubes well the filters were washed with 5 X 3 ml of the latter solution at 2°C, dried and the associated radioactivity was determined in a liquid scintillation spectrometer to measure amsacrine-induced topoisomerase II-DNA complex formation. To measure topoisomerase II activity in isolated nuclei 1 • 105 radioactive nuclei were incubated with or without 10 ~ M amsacrine for 10 min at 37°C in a solution containing 50 mM Tris-HC1 (pH 7.5), 10 mM MgC1 z, 0.12M KCI, 2.25 mM Na EDTA, 5 mM Na + EDTA, 0.5 mM dithiothreitol, 30 # g / m l BSA, 2 mM ATP and adjusted to 50 pA with 25 mM Tris-HC1 (pH 7.5), 35 mM NaC1, 5 mM KCI. Cytoplasmic extracts (usually 30 /~g protein) were added prior to the addition of amsacrine). The nuclei were recovered by centrifugation, resuspended in 1 ml of 137 mM NaCl at 37°C using a vortex mixer then assayed for topoisomerase II activity by co-precipitation with K + / S D S as described above. Results
Effects of serum source on topoisomerase II-DNA complex formation Using the improved conditions for measuring topoisomerase II-DNA complex (PDC) formation described in Materials and Methods we routinely obtained about a 30-fold stimulation of PDC formation when intact K21 or P815 cells were incubated with 10 ~ M amsacrine (Fig. 1A and B). Previously, we also obtained a substantial increase (approx. 10-fold) in PDC formation when isolated K21 cell nuclei were incubated with 10 p~M amsacrine and cytoplasmic extracts from K21 cells [3] and this was confirmed and improved to a 20-25 fold increase using the modified method for isolating K21 cell nuclei described here (Fig. 2). It was also confirmed that 10 p.M amsacrine produced maximum PDC formation by isolated nuclei with cytoplasmic extracts (30 /~g of protein) from cells grown in horse serum (data not shown; cf. Ref. 3). Reasons for the decreased stimulation at higher amsacrine concentrations (Fig. 2) were not determined but it could result from increased intercalation of amsacrine into DNA interfering with topoisomerase action at high drug concentrations. In contrast to these results, when nuclei from P815 cells were incubated with cytoplasmic ex-
261
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Omcentratima of A m s a c r i n e (JAM)
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Fig. 1. PDC formation in intact P815 and K21 cells. (A) The effect of amsacrine concentration on PDC formation. P815 and K2.1 cells were incubated for 30 min with or without increasing amounts of amsacrine and the resulting increase in PDC formation was measured. II, P815 cells; A, K21 cells. (B) The effect of incubation time on PDC formation. P815 and K21 cells were incubated with or without 10 tzM amsacrine for increasing periods and the effect upon PDC formation was measured. II, P815 cells; A, K21 cells. All values are means of three independent experiments, rounded to the nearest whole number.
tracts from P815 cells and 10 tzM amsacrine no stimulation of P D C formation was obtained. This was unexpected because K21 cells are a clonally derived line of P815 cells and there was no difference in the response of the intact cells to amsacrine. For historical reasons our K21 ceils were normally cultured with horse serum whereas P815 ceils were routinely grown in neonatal calf serum, therefore we investigated the possibility that the type of serum used to grow the cells was responsible for the failure of cytoplasmic extracts and nuclei isolated from P815 cells to respond to amsacrine. Overnight cultures of each cell line were established in 2.5% calf serum and in
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2.5% horse serum and PDC formation by the intact cells in response to amsacrine was measured. Both intact K21 and P815 cells produced approx. 30-fold stimulation of PDC formation with amsacrine irrespective of the serum used to grow the cells. However, when different combinations of nuclei and cytoplasmic extracts prepared from the ceils were incubated with 10 /xM amsacrine only cytoplasmic extracts from cells grown with horse serum produced increased PDC formation (20-25-fold) in response to amsacrine (Fig. 3). Because nuclei from P815 cells grown in calf serum responded to amsacrine when provided with cytoplasmic extracts from K21 or P815 ceils grown in horse serum, we concluded that topoisomerase II was not lost during nuclei preparation and that growth in calf serum inhibited or negated the action of the cytoplasmic factor that stimulated topoisomerase 1I in the isolated nuclei. However, the precise reasons were not identified. Instead horse serum was used to grow K21 cells and investigate the effect of arresting the cells by serum deprivation on their ability to respond to amsacrine.
Effects of growth arrest on topoisomerase H activity
0 0
, 10
, 20
, 80
," 40
C,cmeeontmticm ~
Amsaerine (ttM) Fig. 2. The effect of amsacrine concentration on PDC formation in intact K21 cells and in isolated K21 nuclei+cytoplasmicextract. II, intact cells; A, nuclei + extract.
Topoisomerase II activity has been reported to change during the cell cycle and decline in quiescent cells [15,16]. To determine the effect of growth arrest on topoisomerase II in K21 cells, cultures were grown in medium containing 0.05% horse serum for various periods before cytoplasmic extracts and nuclei were prepared from the cells. The nuclei were then incubated with amsacrine and cytoplasmic extracts from log phase cells growing in 2.5% horse serum in order to
262 Cvtoolasmie
Extract
From: 28
K21 n.C.S.
K21 h.s.
0-3
20-25
0-3
20-25
0-3
20-25
N.A.
N.A.
N.A.
20-25
0-3
20-25
¢
P815 rt-C.S.
Time ( h )
• Z
P815 h.s.
0-3
N.A.
0-3
20-25
Data Show fold increase in PDC formation in response to Amsacrine. K21 h.s.
= K21 cells grown in horse serum
P815 n.e.s = P815 cells grown in neonatal calf serum N.A.
Fig. 5. The effect of serum deprivation on PDC forming ability of isolated K21 cell nuclei. The nuclei were incubated with cytoplasmic extract from K21 cells grown in 2.5% horse serum. (©), nuclei from cells grown in 2.5% horse serum; ( • ) , nuclei from cells deprived of serum for increasing times.
= Not assayed
Fig. 3. The effect of culturing cells with calf or horse serum on the ability of cytoplasmic extracts to stimulate PDC formation by isolated nuclei in response to 10 ~ M amsacrine.
detect effects of serum deprivation on topoisomerase II in the nuclei. Cytoplasmic extracts from the serumdeprived cells were also added to nuclei isolated from log phase cells grown in horse serum to detect possible effects of serum deprivation on the topoisomerase IIstimulating factor in the extracts. Fig. 4A illustrates the effect of low serum on growth of the cells and Fig. 4B
shows the effect of serum deprivation on PDC formation by intact ceils in response to amsacrine. There was an almost complete loss of PDC formation after depriving the cells of serum for 12-14 h. In earlier studies we showed by cytofluorimetry that the majority of serum-deprived cells arrest in G1 phase after 12-14 h [17]. When nuclei from serum-deprived or log phase K21 cells were incubated with cytoplasmic extract from log phase cells with or without 10/zM amsacrine and PDC formation was measured there was no decrease in topoisomerase II in the nuclei from growth arrested cells (Fig. 5). However, when cytoplasmic extracts from ceils deprived of serum for various times were added
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8
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T i m e 0a)
.
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20
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2
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T i m e (h)
Fig. 4. The effect of serum concentration on growth and PDC formation by K21 cells. (A) (m), K21 cells grown in 2.5% horse serum; (©), K21 cells grown in 0% horse serum. These cells increasingly lost viability over time. ( • ) , K21 cells grown in 0.05% serum. These cells maintained their viability over 24 h. (B) ( o ) Amsacrine-induced P D C formation by K21 cells grown with horse 2.5% serum; (11), amsacrine-induced PDC formation by K21 cells grown with 0.05% of serum for various times.
263
Time (!1) Fig. 6. The effect of serum deprivation on the ability of cytoplasmic extracts to stimulate P D C formation in isolated nuclei. Cytoplasmic extracts prepared from K21 cells grown in 0.05% serum for increasing periods were added to nuclei from K21 cells grown in 2.5% horse serum and PDC formation in response to 10 tzM amsaerine was determined. (©), cytoplasmic extract from cells in 2.5% serum; ( • ) , cytoplasmic extract from cells in 0.05% serum.
back to nuclei from log phase ceils and PDC formation in response to amsacrine was measured, there was a steady decline in PDC formation with increasing time of serum deprivation as occurred with intact cells (Fig. 6). Cytoplasmic extracts from log phase cells growing with horse serum produced no change in PDC formation with amsacrine over the same period. These results confirmed that the reduction in DNA-topoisomerase II-DNA complex formation in growtharrested K21 cells resulted from decreased activity of the cytoplasmic topoisomerase II-stimulating factor and not decreased topoisomerase II. Discussion
Previously we found that arresting the growth of a cold-sensitive cell cycle mutant of murine mastocytoma cells substantially reduced the topoisomerase II activity in the cells in response to amsacrine and the sensitivity of the cells to the drug [18]. Moreover, inhibitors of RNA or protein synthesis protected the cells from the cytotoxic action of amsacrine without decreasing topoisomerase II activity in nuclear extracts prepared from the cells [19]. This data suggested that an additional growth related labile protein factor was involved in the action of amsacrine on topoisomerase 1I. Further studies showed that cytoplasmic extracts from mastocytoma cells contained a protein factor that enhanced formation of amsacrine-induced topoisomerase II-DNA complexes when added to isolated K21-cell nuclei. No topoisomerase II activity was detectable in the cytoplasmic extracts and the properties of the cytoplasmic factor were not consistent with it being topoisomerase
I or I1 [3]. The cytoplasmic stimulatory factor was eventually isolated, from SDS-PAGE gels, renatured and identified as a 70 kDa protein kinase with properties reminiscent of type II casein kinase. However, the precise identity of the kinase was not determined [4]. The present results confirm the existence of a topoisomerase II-stimulatory factor in mastocytoma ceils and they show that the activity or function of the factor in cell extracts is affected by the type of serum used to grow the cells. Disruption of cells grown with calf serum appeared to inactivate the factor or reverse its action during the preparation of cytoplasmic extracts. Precisely why cytoplasmic extracts from mastocytoma ceils grown in calf serum failed to stimulate topoisomerase II-DNA complex formation in response to amsacrine is unclear. However, since topoisomerase II-DNA complexes were produced by intact cells grown in calf serum, disruption of the ceils appears to be responsible for the loss of cytoplasmic activity and this presumably reflects the prior action of different growth factors or other components in calf or horse serum on intracellular processes. Ericson and Yang [20] have recently described differences between horse and calf serum involving an inhibitor of tumor cell growth. The activity of the topoisomerase II stimulatory factor in cells grown with horse serum was also reduced in extracts from serum-deprived growth arrested cells consistent with a link between growth and topoisomerase II activity. Preliminary evidence suggesting that the stimulatory factor is a protein kinase [4] could be consistent with the model that growth-factors in serum initiate a protein kinase cascade which activates topoisomerase II along with other growth related proteins [21]. Other evidence showing that a casein kinase II type enzyme phosphorylates topoisomerase II in vivo [5], that casein kinase II is a growth-related enzyme [21] and that a casein kinase II-type enzyme phosphorylates and fully restores the DNA cleavage activity of dephosphorylated yeast topoisomerase II [22,23] also suggests that casein kinase II may be the actual entity involved. There was no indication that the level of topoisomerase II was reduced in nuclei from serum-deprived growth-arrested mastocytoma cells, therefore some reports of absence, or changes in topoisomerase II during growth of other ceils may also result from changes in topoisomerase II-stimulatory factors rather than effects upon topoisomerase II itself. Hsiang et al [24] found little change in topoisomerase I! in various other tumor cells under different growth conditions. Our earlier results with cycloheximide or cordycepin suggested that continuous protein and RNA synthesis are necessary to maintain the activity of topoisomerase II in mastocytoma cells [3]. Therefore, loss of stimulatory activity upon serum-deprivation or growth arrest could result from decreased synthesis or increased
264 turnover of the cytoplasmic factor. However, whether the factor, or its activity is dependent upon continuous protein synthesis or other factors was not resolved. It is important to understand these processes as they appear to control the response of cells to drugs such as amsacrine and they could be implicated in the resistance of non-cycling tumour cells to anti-cancer drugs. Finally, most cells are grown with calf serum which may obscure the topoisomerase ll-stimulatory factor in other in vitro cell-free systems.
Acknowledgements This research was supported by grants from the Auckland Division, Cancer Society of New Zealand, and the New Zealand Lottery Board. References 1 Liu, L.F. (1989) Annu. Rev. Biochem. 58, 351-375. 2 Rowe, T.C., Chen, G.L., Hsiang, Y.H. and Liu, L.F. (1986) Cancer Res. 46, 2021-2026. 3 Darkin, S.J. and Ralph, R.K. (1989) Biochim. Biophys. Acta 1007, 295 -300. 4 Darkin-Ranray, S.J. and Ralph, R.K. (1991) Biochim. Biophys. Acta 1088, 285-291. 5 Ackermann, P., Glover, C.V.C. and Osheroff, N. (1988) J. Biol. Chem. 263, 12653-12660. 6 Saijo, M., Enomoto, T., Hanaoka, F. and Ui, M. (1990) Biochemistry 29, 583-590.
7 Constantinou, A., Henning-Chub, C. and Huberman, E. (1989) Cancer Res. 49, 111{)-1117. 8 Takano, H., Kohno, K., Ono, M., Uchida, Y. and Kuwano, M. (1991) Cancer Res. 51, 3951 3957, 9 Darby, M.K., Schmitt, J., Jongstra-Bilen, J. and Vosberg, H.P. (1985) EMBO J. 4, 2129-2134. 10 Chow, K.C., King, C.K. and Ross, W.E. (1988) Biochem. Pharmacol. 37, 1117-1122 11 Fry, A.M., Chresta, C.M., Davies, S.M., Walker, M.C., Harris, A.L., Hartley, J.A., Masters, J.R.W. and Hickson, I.D. (1991) Cancer Res. 51, 6592-6595. 12 De Isabella, P., Capranico, G., Binaschi, M., Tinelli, S. and Zunino, F. (1990) Mol. Pharmacol. 37 11-16. 13 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 14 Blank-Liss, W. and Schindler, R. (1985) Biochim. Biophys. Acta. 826, 213-223. 15 Hsiang, Y.-H., Wu, H-Y. and Liu, L.F. (1988) Cancer Res. 48, 3230-3235. 16 Heck, M.M.S., Hittelman, W.N. and Earnshaw, W.C. (1988) Proc. Natl. Acad. Sci. USA 85, 1086-1090. 17 Goulding, M. and Ralph, R.K. (1985) Mol. Cell Biochem. 67, 31-38. 18 Schneider, E., Hutchins, A.-M., Darkin, S.J., Lawson, P.A. and Ralph, R.K. (1988) Biochim. Biophys. Acta. 951, 85-97. 19 Schneider, E., Lawson, P.A. and Ralph, R.K. (1989) Biochem. Pharmacol. 38, 263-269. 20 Ericson, K.K. and Yang, T.J. (1992) In Vitro 28A, 11-16. 21 Ralph, R.K., Darkin-Rattray, S. and Schofield, P. (1990) Bioessays 12, 121-124. 22 Cardenas, M. and Alghisi, C. (1991) Annual Report Swiss Cancer Resarch Institute, pp. 77-78. 23 Cardenas, M.E., Dang, Q., Glover, C.V.C. and Gasser, S.M. (1992) EMBO J. 11, 1785-1796. 23 Hsiang, Y-H., Wu, H.Y. and Liu, L.F. (1988) Cancer Res. 48, 3230-3235.
259
BBAEXP 92418
Regulation of topoisomerase II by murine mastocytoma cells Andrew G. Collett and Ray K. Ralph Department of Cellular and Molecular Biology, Unit:ersityof Auckland, Auckland (New Zealand) (Received 24 April 1992)
Key words: Topoisomerase II; Growth; Cytoplasmic factor; (Mouse)
Nuclei from K21 murine mastocytoma cells do not form topoisomerase II-DNA adducts in response to amsacrine in the absence of a cytoplasmic factor tentatively identified as a type of casein kinase (Darkin, S.J. and Ralph, R.K. (1991) Biochim. Biophys. Acta 1088, 285-291). The stimulatory activity was present in extracts from cells grown in horse serum but not in calf serum. Activity was lost following growth arrest by serum deprivation. In contrast, topoisomerase II activity in isolated nuclei did not decline during growth arrest. These results suggest that the resistance of some non-cycling tumour cells to anti-cancer drugs may result from decreased activation of topoisomerase II.
Introduction
D N A topoisomerase II is the nuclear target for a variety of DNA-intercalating anticancer drugs, including the 9-anilino acridine derivative amsacrine [1]. Amsacrine prevents the religation of D N A during the DNA cleavage-resealing reaction catalysed by topoisomerase II and the resulting covalently-linked complexes between DNA and topoisomerase II can be precipitated with K+-sodium dodecylsulphate (SDS) and quantified to measure the activity of the enzyme [2]. Using this technique we demonstrated that isolated nuclei from K21 murine mastocytoma cells would not form topoisomerase II-DNA adducts in response to amsacrine and that a cytoplasmic factor in the ceils facilitated the formation of amsacrine-induced topoisomerase II-DNA complexes (PDC) when cytoplasmic extracts were added to isolated nuclei in vitro [3]. Evidence was also presented that the cytoplasmic factor involved was a 70 kDa protein kinase with properties reminiscent of casein kinase II [4]. Other data also suggest that phosphorylation [5-8], ADP-ribosylation [9], or unidentified factors [10-12] may regulate the activity of topoisomerase II. While investigating the universality and properties of the cytoplasmic factor that activates topoisomerase II in isolated nuclei from mastocytoma cells, we found
Correspondence to: R.K. Ralph, Department of Cellular and Molecular Biology, University of Auckland, Private Bag 92019, Auckland, New Zealand. Abbreviations: SDS, sodium dodecylsulphate; PDC, protein-DNA complexes.
that cytoplasmic extracts from the parent P815 clone of mastocytoma cells did not increase the formation of DNA-topoisomerase II complexes in isolated nuclei treated with amsacrine. Here we show that the different effects of cytoplasmic extracts from K21 and P815 cells are a result of the different serum used to culture the two clones of cells, and that no stimulatory activity is detectable when cytoplasmic extracts are prepared from K21 or P815 ceils grown with neonatal calf serum. Effects of growth arrest by serum deprivation on topoisomerase II in nuclei, and the topoisomerase 1I stimulatory activity in cytoplasmic extracts from K21 mastocytoma ceils grown in horse serum are also described. The results suggest that factors in horse serum induce or activate the cytoplasmic factor (kinase) in mastocytoma cells that is essential for topoisomerase II to respond to amsacrine. Materials and Methods
General methods Mastocytoma cells were grown in suspension in RPMI1640 medium with either neonatal calf serum or horse serum [4], both products of Gibco-BRL (NZ). Cell density was determined with a Neubauer haemocytometer and cell viability by trypan blue exclusion. Proteinase inhibitors used to prepare cytoplasmic extracts were obtained from Boehringer-Mannheim or Sigma and solutions prepared immediately before use. Protein concentrations were measured using Bradford's reagent [13]. During nuclei isolation cell lysis was confirmed by staining nuclei with aniline blue (0.1%, w / v ) dissolved in nuclei isolation buffer (see below).
260 To radiolabel DNA log phase cells (2. l05 cells/ml) were grown with 0.5/~Ci/ml [methyl-3H]thymidine (20 C i / m m o l ) and 1 ~ M thymidine for 16 h. The cells contained approx. 30000 c p m / 1 " 105 cells after precipitation with trichloroacetic acid, collection of the precipitates on G F / C glass fiber filters and measurement in a liquid scintillation spectrometer.
Cytoplasmic extracts Cytoplasmic extracts were prepared from 5 • 108 cells using a slight modification of the procedure of BlankLiss and Schindler [14]. The cells were recovered by centrifugation at 900 x g for 10 min at 2°C, washed twice with ice-cold Tris-buffered saline and the final pellet was resuspended in 17 mM Mops (pH 7), 250 mM sucrose, 2.5 mM EDTA, 400 K I U / m l aprotinin, 0.1 m g / m l leupeptin, 0.01 m g / m l a-macroglobulin, 1 mM phenylmethylsulphonyl fluoride~ 0.1 m g / m l diisopropylfluorophosphate, 2 m g / m l digitonin at 20°C and a final density of 1.2.108 cells/ml. After 10 min at 20°C the lysate was centrifuged at 30000 x g for 10 min at 0°C, the supernatant was recovered, diluted to 4 m g / m l protein and stored as 0.1 ml aliquots at -70°C. Nuclei isolation Nuclei were prepared from 1 - 108 cells pre-labeled with [3H]thymidine. The cells were collected by centrifugation at 1600 x g for 5 min and resuspended in 2 ml of 20 mM Tris-HCl (pH 7.2), 150 mM KC1, 5 mM MgCI 2, 10 mM NazS205, 2% (w/v), Dextran 150-200 then mixed with 2 ml of the same buffer containing (I.1% Triton X-100 in a tight Dounce homogenizer. After four u p / d o w n strokes of the pestle the mixture was kept on ice for 10 rain with occasional gentle vortex mixing. The nuclei were collected by centrifugation at 1 2 0 0 x g for 10 min at 2°C, resuspended to 6" 10 7 nuclei/ml in the nuclei isolation buffer and used immediately. Topoisomerase H assays To measure topoisomerase II activity, intact cells labeled with [3H]thymidine were recovered by centrifugation, washed twice at 37°C with medium containing the same serum used to grow the cells, resuspended in the same medium at 2.105 cells/ml and 1 ml aliquots were distributed into 1.5 ml Eppendorf microfuge tubes at 37°C. The cells were first incubated for 30 min at 37°C then with or without 10 /~M amsacrine for a further 10 min. After centrifugation the cells were resuspended in 1 mi 137 mM NaC1 at 37°C using a vortex mixer and lysed by adding 0.1 ml 12.5% (w/v) SDS, 50 mM E D T A (Na+), 4 m g / m l salmon sperm DNA prewarmed to 65°C. This mixture was gently sucked up and down three times in a siliconized pasteur pipette then incubated at 65°C for 3 min before adding 0.25 ml of 325 mM KCI at 37°C to each tube
and immediately mixing the solutions for l0 s using a vortex mixer at its highest setting. After chilling the tubes on ice for 40 rain the precipitates that formed were centrifuged at 15000 × g for 10 min at 4°C, the pellets were resuspended in 1 ml 10 mM Tris-HC1 (pH 7.5), 100 mM KCI, 5 mM EDTA, 2 p~g/ml salmon sperm DNA, 1 p.M thymidine at 4°C, dispersed using a siliconized pasteur pipette, and collected under gravity on G F / C glass fiber filters presoaked in 5 ~ g / m l salmon sperm DNA. After rinsing the tubes well the filters were washed with 5 X 3 ml of the latter solution at 2°C, dried and the associated radioactivity was determined in a liquid scintillation spectrometer to measure amsacrine-induced topoisomerase II-DNA complex formation. To measure topoisomerase II activity in isolated nuclei 1 • 105 radioactive nuclei were incubated with or without 10 ~ M amsacrine for 10 min at 37°C in a solution containing 50 mM Tris-HC1 (pH 7.5), 10 mM MgC1 z, 0.12M KCI, 2.25 mM Na EDTA, 5 mM Na + EDTA, 0.5 mM dithiothreitol, 30 # g / m l BSA, 2 mM ATP and adjusted to 50 pA with 25 mM Tris-HC1 (pH 7.5), 35 mM NaC1, 5 mM KCI. Cytoplasmic extracts (usually 30 /~g protein) were added prior to the addition of amsacrine). The nuclei were recovered by centrifugation, resuspended in 1 ml of 137 mM NaCl at 37°C using a vortex mixer then assayed for topoisomerase II activity by co-precipitation with K + / S D S as described above. Results
Effects of serum source on topoisomerase II-DNA complex formation Using the improved conditions for measuring topoisomerase II-DNA complex (PDC) formation described in Materials and Methods we routinely obtained about a 30-fold stimulation of PDC formation when intact K21 or P815 cells were incubated with 10 ~ M amsacrine (Fig. 1A and B). Previously, we also obtained a substantial increase (approx. 10-fold) in PDC formation when isolated K21 cell nuclei were incubated with 10 p~M amsacrine and cytoplasmic extracts from K21 cells [3] and this was confirmed and improved to a 20-25 fold increase using the modified method for isolating K21 cell nuclei described here (Fig. 2). It was also confirmed that 10 p.M amsacrine produced maximum PDC formation by isolated nuclei with cytoplasmic extracts (30 /~g of protein) from cells grown in horse serum (data not shown; cf. Ref. 3). Reasons for the decreased stimulation at higher amsacrine concentrations (Fig. 2) were not determined but it could result from increased intercalation of amsacrine into DNA interfering with topoisomerase action at high drug concentrations. In contrast to these results, when nuclei from P815 cells were incubated with cytoplasmic ex-
261
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Omcentratima of A m s a c r i n e (JAM)
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Fig. 1. PDC formation in intact P815 and K21 cells. (A) The effect of amsacrine concentration on PDC formation. P815 and K2.1 cells were incubated for 30 min with or without increasing amounts of amsacrine and the resulting increase in PDC formation was measured. II, P815 cells; A, K21 cells. (B) The effect of incubation time on PDC formation. P815 and K21 cells were incubated with or without 10 tzM amsacrine for increasing periods and the effect upon PDC formation was measured. II, P815 cells; A, K21 cells. All values are means of three independent experiments, rounded to the nearest whole number.
tracts from P815 cells and 10 tzM amsacrine no stimulation of P D C formation was obtained. This was unexpected because K21 cells are a clonally derived line of P815 cells and there was no difference in the response of the intact cells to amsacrine. For historical reasons our K21 ceils were normally cultured with horse serum whereas P815 ceils were routinely grown in neonatal calf serum, therefore we investigated the possibility that the type of serum used to grow the cells was responsible for the failure of cytoplasmic extracts and nuclei isolated from P815 cells to respond to amsacrine. Overnight cultures of each cell line were established in 2.5% calf serum and in
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2.5% horse serum and PDC formation by the intact cells in response to amsacrine was measured. Both intact K21 and P815 cells produced approx. 30-fold stimulation of PDC formation with amsacrine irrespective of the serum used to grow the cells. However, when different combinations of nuclei and cytoplasmic extracts prepared from the ceils were incubated with 10 /xM amsacrine only cytoplasmic extracts from cells grown with horse serum produced increased PDC formation (20-25-fold) in response to amsacrine (Fig. 3). Because nuclei from P815 cells grown in calf serum responded to amsacrine when provided with cytoplasmic extracts from K21 or P815 ceils grown in horse serum, we concluded that topoisomerase II was not lost during nuclei preparation and that growth in calf serum inhibited or negated the action of the cytoplasmic factor that stimulated topoisomerase 1I in the isolated nuclei. However, the precise reasons were not identified. Instead horse serum was used to grow K21 cells and investigate the effect of arresting the cells by serum deprivation on their ability to respond to amsacrine.
Effects of growth arrest on topoisomerase H activity
0 0
, 10
, 20
, 80
," 40
C,cmeeontmticm ~
Amsaerine (ttM) Fig. 2. The effect of amsacrine concentration on PDC formation in intact K21 cells and in isolated K21 nuclei+cytoplasmicextract. II, intact cells; A, nuclei + extract.
Topoisomerase II activity has been reported to change during the cell cycle and decline in quiescent cells [15,16]. To determine the effect of growth arrest on topoisomerase II in K21 cells, cultures were grown in medium containing 0.05% horse serum for various periods before cytoplasmic extracts and nuclei were prepared from the cells. The nuclei were then incubated with amsacrine and cytoplasmic extracts from log phase cells growing in 2.5% horse serum in order to
262 Cvtoolasmie
Extract
From: 28
K21 n.C.S.
K21 h.s.
0-3
20-25
0-3
20-25
0-3
20-25
N.A.
N.A.
N.A.
20-25
0-3
20-25
¢
P815 rt-C.S.
Time ( h )
• Z
P815 h.s.
0-3
N.A.
0-3
20-25
Data Show fold increase in PDC formation in response to Amsacrine. K21 h.s.
= K21 cells grown in horse serum
P815 n.e.s = P815 cells grown in neonatal calf serum N.A.
Fig. 5. The effect of serum deprivation on PDC forming ability of isolated K21 cell nuclei. The nuclei were incubated with cytoplasmic extract from K21 cells grown in 2.5% horse serum. (©), nuclei from cells grown in 2.5% horse serum; ( • ) , nuclei from cells deprived of serum for increasing times.
= Not assayed
Fig. 3. The effect of culturing cells with calf or horse serum on the ability of cytoplasmic extracts to stimulate PDC formation by isolated nuclei in response to 10 ~ M amsacrine.
detect effects of serum deprivation on topoisomerase II in the nuclei. Cytoplasmic extracts from the serumdeprived cells were also added to nuclei isolated from log phase cells grown in horse serum to detect possible effects of serum deprivation on the topoisomerase IIstimulating factor in the extracts. Fig. 4A illustrates the effect of low serum on growth of the cells and Fig. 4B
shows the effect of serum deprivation on PDC formation by intact ceils in response to amsacrine. There was an almost complete loss of PDC formation after depriving the cells of serum for 12-14 h. In earlier studies we showed by cytofluorimetry that the majority of serum-deprived cells arrest in G1 phase after 12-14 h [17]. When nuclei from serum-deprived or log phase K21 cells were incubated with cytoplasmic extract from log phase cells with or without 10/zM amsacrine and PDC formation was measured there was no decrease in topoisomerase II in the nuclei from growth arrested cells (Fig. 5). However, when cytoplasmic extracts from ceils deprived of serum for various times were added
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0
.
4
.
.
8
.
.
12
T i m e 0a)
.
16
0
20
24
2
4
6
8
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12
T i m e (h)
Fig. 4. The effect of serum concentration on growth and PDC formation by K21 cells. (A) (m), K21 cells grown in 2.5% horse serum; (©), K21 cells grown in 0% horse serum. These cells increasingly lost viability over time. ( • ) , K21 cells grown in 0.05% serum. These cells maintained their viability over 24 h. (B) ( o ) Amsacrine-induced P D C formation by K21 cells grown with horse 2.5% serum; (11), amsacrine-induced PDC formation by K21 cells grown with 0.05% of serum for various times.
263
Time (!1) Fig. 6. The effect of serum deprivation on the ability of cytoplasmic extracts to stimulate P D C formation in isolated nuclei. Cytoplasmic extracts prepared from K21 cells grown in 0.05% serum for increasing periods were added to nuclei from K21 cells grown in 2.5% horse serum and PDC formation in response to 10 tzM amsaerine was determined. (©), cytoplasmic extract from cells in 2.5% serum; ( • ) , cytoplasmic extract from cells in 0.05% serum.
back to nuclei from log phase ceils and PDC formation in response to amsacrine was measured, there was a steady decline in PDC formation with increasing time of serum deprivation as occurred with intact cells (Fig. 6). Cytoplasmic extracts from log phase cells growing with horse serum produced no change in PDC formation with amsacrine over the same period. These results confirmed that the reduction in DNA-topoisomerase II-DNA complex formation in growtharrested K21 cells resulted from decreased activity of the cytoplasmic topoisomerase II-stimulating factor and not decreased topoisomerase II. Discussion
Previously we found that arresting the growth of a cold-sensitive cell cycle mutant of murine mastocytoma cells substantially reduced the topoisomerase II activity in the cells in response to amsacrine and the sensitivity of the cells to the drug [18]. Moreover, inhibitors of RNA or protein synthesis protected the cells from the cytotoxic action of amsacrine without decreasing topoisomerase II activity in nuclear extracts prepared from the cells [19]. This data suggested that an additional growth related labile protein factor was involved in the action of amsacrine on topoisomerase 1I. Further studies showed that cytoplasmic extracts from mastocytoma cells contained a protein factor that enhanced formation of amsacrine-induced topoisomerase II-DNA complexes when added to isolated K21-cell nuclei. No topoisomerase II activity was detectable in the cytoplasmic extracts and the properties of the cytoplasmic factor were not consistent with it being topoisomerase
I or I1 [3]. The cytoplasmic stimulatory factor was eventually isolated, from SDS-PAGE gels, renatured and identified as a 70 kDa protein kinase with properties reminiscent of type II casein kinase. However, the precise identity of the kinase was not determined [4]. The present results confirm the existence of a topoisomerase II-stimulatory factor in mastocytoma ceils and they show that the activity or function of the factor in cell extracts is affected by the type of serum used to grow the cells. Disruption of cells grown with calf serum appeared to inactivate the factor or reverse its action during the preparation of cytoplasmic extracts. Precisely why cytoplasmic extracts from mastocytoma ceils grown in calf serum failed to stimulate topoisomerase II-DNA complex formation in response to amsacrine is unclear. However, since topoisomerase II-DNA complexes were produced by intact cells grown in calf serum, disruption of the ceils appears to be responsible for the loss of cytoplasmic activity and this presumably reflects the prior action of different growth factors or other components in calf or horse serum on intracellular processes. Ericson and Yang [20] have recently described differences between horse and calf serum involving an inhibitor of tumor cell growth. The activity of the topoisomerase II stimulatory factor in cells grown with horse serum was also reduced in extracts from serum-deprived growth arrested cells consistent with a link between growth and topoisomerase II activity. Preliminary evidence suggesting that the stimulatory factor is a protein kinase [4] could be consistent with the model that growth-factors in serum initiate a protein kinase cascade which activates topoisomerase II along with other growth related proteins [21]. Other evidence showing that a casein kinase II type enzyme phosphorylates topoisomerase II in vivo [5], that casein kinase II is a growth-related enzyme [21] and that a casein kinase II-type enzyme phosphorylates and fully restores the DNA cleavage activity of dephosphorylated yeast topoisomerase II [22,23] also suggests that casein kinase II may be the actual entity involved. There was no indication that the level of topoisomerase II was reduced in nuclei from serum-deprived growth-arrested mastocytoma cells, therefore some reports of absence, or changes in topoisomerase II during growth of other ceils may also result from changes in topoisomerase II-stimulatory factors rather than effects upon topoisomerase II itself. Hsiang et al [24] found little change in topoisomerase I! in various other tumor cells under different growth conditions. Our earlier results with cycloheximide or cordycepin suggested that continuous protein and RNA synthesis are necessary to maintain the activity of topoisomerase II in mastocytoma cells [3]. Therefore, loss of stimulatory activity upon serum-deprivation or growth arrest could result from decreased synthesis or increased
264 turnover of the cytoplasmic factor. However, whether the factor, or its activity is dependent upon continuous protein synthesis or other factors was not resolved. It is important to understand these processes as they appear to control the response of cells to drugs such as amsacrine and they could be implicated in the resistance of non-cycling tumour cells to anti-cancer drugs. Finally, most cells are grown with calf serum which may obscure the topoisomerase ll-stimulatory factor in other in vitro cell-free systems.
Acknowledgements This research was supported by grants from the Auckland Division, Cancer Society of New Zealand, and the New Zealand Lottery Board. References 1 Liu, L.F. (1989) Annu. Rev. Biochem. 58, 351-375. 2 Rowe, T.C., Chen, G.L., Hsiang, Y.H. and Liu, L.F. (1986) Cancer Res. 46, 2021-2026. 3 Darkin, S.J. and Ralph, R.K. (1989) Biochim. Biophys. Acta 1007, 295 -300. 4 Darkin-Ranray, S.J. and Ralph, R.K. (1991) Biochim. Biophys. Acta 1088, 285-291. 5 Ackermann, P., Glover, C.V.C. and Osheroff, N. (1988) J. Biol. Chem. 263, 12653-12660. 6 Saijo, M., Enomoto, T., Hanaoka, F. and Ui, M. (1990) Biochemistry 29, 583-590.
7 Constantinou, A., Henning-Chub, C. and Huberman, E. (1989) Cancer Res. 49, 111{)-1117. 8 Takano, H., Kohno, K., Ono, M., Uchida, Y. and Kuwano, M. (1991) Cancer Res. 51, 3951 3957, 9 Darby, M.K., Schmitt, J., Jongstra-Bilen, J. and Vosberg, H.P. (1985) EMBO J. 4, 2129-2134. 10 Chow, K.C., King, C.K. and Ross, W.E. (1988) Biochem. Pharmacol. 37, 1117-1122 11 Fry, A.M., Chresta, C.M., Davies, S.M., Walker, M.C., Harris, A.L., Hartley, J.A., Masters, J.R.W. and Hickson, I.D. (1991) Cancer Res. 51, 6592-6595. 12 De Isabella, P., Capranico, G., Binaschi, M., Tinelli, S. and Zunino, F. (1990) Mol. Pharmacol. 37 11-16. 13 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 14 Blank-Liss, W. and Schindler, R. (1985) Biochim. Biophys. Acta. 826, 213-223. 15 Hsiang, Y.-H., Wu, H-Y. and Liu, L.F. (1988) Cancer Res. 48, 3230-3235. 16 Heck, M.M.S., Hittelman, W.N. and Earnshaw, W.C. (1988) Proc. Natl. Acad. Sci. USA 85, 1086-1090. 17 Goulding, M. and Ralph, R.K. (1985) Mol. Cell Biochem. 67, 31-38. 18 Schneider, E., Hutchins, A.-M., Darkin, S.J., Lawson, P.A. and Ralph, R.K. (1988) Biochim. Biophys. Acta. 951, 85-97. 19 Schneider, E., Lawson, P.A. and Ralph, R.K. (1989) Biochem. Pharmacol. 38, 263-269. 20 Ericson, K.K. and Yang, T.J. (1992) In Vitro 28A, 11-16. 21 Ralph, R.K., Darkin-Rattray, S. and Schofield, P. (1990) Bioessays 12, 121-124. 22 Cardenas, M. and Alghisi, C. (1991) Annual Report Swiss Cancer Resarch Institute, pp. 77-78. 23 Cardenas, M.E., Dang, Q., Glover, C.V.C. and Gasser, S.M. (1992) EMBO J. 11, 1785-1796. 23 Hsiang, Y-H., Wu, H.Y. and Liu, L.F. (1988) Cancer Res. 48, 3230-3235.
