Enhancement of X-Ray Cell Killing in Cultured Mammalian Cells by the Protein Phosphatase Inhibitor Calyculin A

[CANCERRESEARCH54,2088-2090, April 15, 19941 Advances in Brief

Katsumasa Nakamura' and Shigetoshi Antoku

Department of Experimental Radiology, Faculty ofMedicine, Kyushu University, Maidashi 3-1-1, Higashi-ku, FU/cuOka,812 Japan

Abstract (13), exponentially growing cells were irradiated at room temperature in suspension, which were trypsinized and suspended in fresh medium (1 X 10@ Effects of calyculin A, a potent unhibutorof protein phosphatases 1 and cells/ml) just before X-irradiation. 2A, on X-ray cell killing and chromatin structure were studied using Colony Survival Assay. Cell survival was determinedusing a conven cultured mammalian cells (BHK21). Calyculin A at concentrations of tional colony-forming assay. After postirradiation treatment with CL-A for 30 2.5-20 flM enhancedX-ray cell killing whenexponentiallygrowing BHK21 mm at 37Â°C,thedrug was removedby two washeswith fresh medium.For cells were treated with calyculin A for 30 mm after X-irradiation. A control survival curves, irradiated cells were incubated in fresh medium for 30 30-mm treatment with this drug induced cbromatin condensation tran min. Subsequently, cells were counted, diluted, and plated in 60-mm dishes. siently. These results suggest that the enhancement ofX-ray cell killing by After incubation for 6 days, the colonies were fixed, stained, and then counted. calyculin A is caused by the events associated with chromatin condensa The platingefficiency of unirradiatedcontrolcells was 75â€”85%.Theresults don. Protein phosphatase-targeting drugs may represent a new class of for CL-A treatment were normalized for the cytotoxic effects of CL-A alone. radiation sensitizers. Inthe figuresto follow, the meanvalue andthe SE, which were obtainedin at least three experiments, are shown. The survival curves were described using Introduction the traditional single-hit, multitarget formula. D0 and Dq values were deter mined by performing a least-squares linear regression analysis from the data Phosphorylation and dephosphorylation of proteins play an impor points on the linear portion of the survival curve. tant role in the control of cellular metabolism, growth, and differen Detection of Morphological Changes in Chromatin. Chromosomal mor tiation in eukaryotic cells. Recent fmdings also suggest that protein phology was evaluated using fluorescence microscopy. At the end of each kinase-mediated regulation is associated with the cellular response to incubation, cells were detached by trypsinizing. Both the floating and ionizing radiation (1â€”4).In particular, several reports have empha trypsinized cells were collected by centrifugation. Cells were then fixed in sized a role for protein kinase C in the repair processes of radiation absolute methanol and stained for 10 mm with 50 @g/mlpropidiumiodide induced lesions (2â€”4). Although the level of phosphorylation of (Sigma Chemical Co., St.Louis, MO). proteins is dependent on the relative activities of protein kinases and Results phosphatases, less attention has been given to the latter enzymes. CL-A2 is a specific inhibitor of protein phosphatases 1 and 2A Phase Contrast Microscopic Observations. When BHK21 cells isolated from the marine sponge Discodermia calyx (5). Interestingly, were treated with 10 n@iCL-A for 30 mm, almost all cells rounded up it has been reported that CL-A induces chromatin condensation in and became detached from the culture dish. These changes were unfertilized sea urchin eggs (6). If CL-A also affects chromatin-DNA reversible. Within 1 h after removal of CL-A, the rounded cells structure in mammalian cells, it would be of interest to determine attached to the substratum and then resumed a flattened shape. The whether CL-A modifies the radiosensitivity of mammalian cells be vast majority of cells were active mitotically (data not shown). cause conformational changes in chromatin are one of the factors Cytotoxicity and Radioenhancement Induced by CL-A. Fig. IA which influence cellular radiosensitivity (7â€”11).In this paper, we shows the effects of 30-min treatments with various concentrations show that CL-A induces chromatin condensation and enhances X-ray (2.5â€”20nM)of CL-A on the survival of BHK21 cells exposed to 4 Gy cell killing in a BHK21 fibroblast line. of X-rays or unexposed to X-rays. Relatively few unirradiated cells were killed, even after treatment with 20 n@iCL-A. However, the Materials and Methods killing of irradiated cells was markedly enhanced by the CL-A treat ment. Fig. lB shows radiation survival curves of BHK21 cells inca Cell Culture. The culturedmammaliancellswere a BHK21fibroblastline bated in the absence or presence of CL-A (2.5, 5.0, or 10 nM) for 30 derived from the Syrian hamster. BHK21 cells were cultured as monolayers in mm after X-irradiation. It is apparent that the magnitude of radiation Dulbecco's modified Eagle's minimum essential medium supplemented with enhancement is directly dependent on the concentration of the drug 10% fetal calf serum and grew exponentially with a doubling time of about after X-irradiation. The D0 and Dq values for each CL-A concentra ii h. Chemicals. CL-A (Wako Pure Chemical Industries, Ltd., Osaka, Japan) tion are presented in Table 1. The D0 declined from 1.94 Gy in was dissolved in ethanolto a stock concentrationof0.5 ELM.Forexperiments, untreated cultures to 0.92 Gy in cultures incubated with 10 nr@iCL-A, appropriate volume of drug-containing solution was added to cultures. while the Dq parameters did not change significantly. Thus, the major Irradiation. The radiation used was 200 kVp X-rays with 0.3 mm copper effect of the drug appeared to be an increase in the slope of the filtration at a dose rate of 1.49 Gy/min. CL-A-treated BHK21 cells rounded up survival curve. and became detached from the culture dish, as shown by Chartier et aL(12). To Premature Chromatin Condensation Induced by CL-A. Mor exclude the influence of morphological changes on cellular radiosensitivity phological changes in chromatin by CL-A are demonstrated in Fig. 2 and Fig. 3A. Three % of untreated control cells contained the mitotic Received 1/10/94; accepted 3/4/94. chromosomes. When BHK21 cells were incubated with 10 nMCL-A, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with cells demonstrated chromatin condensation with collapsed nuclei (Fig. 18 U.S.C. Section 1734 solely to indicate this fact. 24). The condensation was completed within 6 h of exposure to CL-A I To whom requests for reprints should be addressed. (Fig. 3A). 2 The abbreviationa used are: CL-A, calyculin A; BIlK, baby hamster kidney; D(,, dose requfredto reducesurvival37% on an exponentialportionof a survivalcurve;Dq, Fig. 3B shows kinetics of chromatin condensation after a 30-usia quasi-threshold dose. treatment with 10 n@iCL-A following X-irradiation. The induction of 2088

1 . -- killing (8, 10). Sasaki and Nishimoto (1 1) extended these studies and

C showed that the lethal effect of X rays was enhanced at a nonpermis @ 0 0 e sive temperature (40Â°C)in a temperature-sensitive mutant of BHK21 U I- cells (tsBN2), which demonstrated premature chromatin condensation at the nonpermissive temperature. Therefore, it is not surprising that 0.1 C CL-A, which induces chromatin condensation, enhances X-ray induced cell killing in BHK21 cells. The level of phosphorylation of specific regulatory proteins is U) A dependent on the relative activities of protein kinases and phospha

Conlr@ 10 100 Concentration (nM)

C . 0 Is 0.1

0.01 U) D A

B 0.001 2 4 6 8 10 Dose (Gy) S Fig. 1. A, effects of various concentrationsof CL-A on the survival of control or irradiatedBHK21cells.CeHswereirradiatedwitheither0 Gy(â€¢)or4 Gy(0) of X-rays andthentreatedwithupto 20natCL-Afor30mis.Thecolonysurvivalassaywasused to determinecellsurvival.Thedataat 4 Gy are normalizedforthe cytotoxiceffectsof CL-A alone. B, radiation survival curves of cells treated with CL-A. Immediately after radiation exposure, cells were incubated with 2.5 nat CL-A (0), 5 nu CL-A (A) or 10 ns@ CL-A (0) for 30 mis. Control cells (â€¢)were incubated without CL-A for 30 min after X-frradiation. The data are normalized for the cytotoxic effects of the drug alone. B Fig. 2. Changes in chromosomal morphology after 30-mm treatment with CL-A. A, 10 flM CL-A B, control. fluorescence micrograph, X 400. CL.ADrugTable 1 Sensitization by 30-mm pOstirTadiatiOn treaintent with

concentration(nat)D0(Gy)@01.94 (Gy)aDg 0.2092.51.65 Â±0.O62@'1.59 Â± 0.4675.01.37 Â±0.1521.65 Â± C 0.49710.00.92 Â±0.1071.40 Â± 0 Â±0.0121.20 Â±0.059

a ii@ and Dq values were determined by performing a least-squares linear regression C analysisasdescribedhiâ€œMaterialsandMethods.â€• S â€˜D M@ Â±SE. C 0 U

chromatin condensation was observed within 30 usia. Even after C removal of CL-A, the induction was seen, and the maximal level of S E chromatin condensation was observed after 1 h of removal of the drug. 0 Subsequently, the condensed chromatin began to decondense again, C.) and the structure of the nucleus reverted to that in untreated cells. In contrast, irradiation alone induced a cessation of mitotic activity, indicative of a period of arrest in G2 (14).

Discussion 0 1 2 3 4 5 6 0123456 Time (h) Chromatin conformation is thought to be one of the important Fug.3. A, kinetics of the unductuonofchromatin condensation by calyculunA. BHK21 factors in determining cellular radiosensitivity. The radiosensitivity cells in suspension were treated with 10 nu calyculin A for various times of incubation. depends on the stage of the cell cycle, and cells are more sensitive to B, kinetics of condensed chromatin after X-irradiation. Immediately after exposure to 4 ionizing radiation at stages of high chromatin condensation (7). Post Gy, BHK21cells in suspensuonwereseeded in the presence(S) or absence(0) of 10 nM CL-A at time 0. After 30 mm, cells were rinsed and plated in the dishes containing fresh irradiation treatment with agents such as hypertonic solution and medium. At least 1000 cells were examined by fluorescence microscopy to determine the caffeine, which induce chromatin condensation, enhances X-ray cell percentageofthecellscontainingmitoticchromosomesorcondensedchromatin. 2089

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. RADIOENHANCEMENTBY CALYCUUN A tases. Thus, the inhibition of protein phosphatases increases phospho References rylation of proteins (12, 15) and may finally activate intracellular 1. Rowley, R., Hudson,J., and Young, P. G. The Weal proteinkinase is requiredfor signaling pathways. In the sea urchin eggs, CL-A activates histone Hi radiation-inducedmitoticdelay.Nature(Land.),356:353â€”355,1992. 2. Woloschak, G. E., Chang-Liu, C., and Shearin-Jones, P. Regulation of protein kinase kinase in a cyclin B-independent manner by blocking the activity of C by ionizing radiation.CancerRes., 50: 3963â€”3967,1990. protein phosphatase 1 (6). Okadaic acid, another specific inhibitor of 3. Hallahan, D. E., Virudachalam, S., Sherman, M. L, Huberman, E., Kufe, D. W., and protein phosphatases 1 and 2A, also activates histone Hi kinase Weichselbaum,R. R. Tumornecrosisfactorgene expressionis mediatedby protein kinase C following activation by ionizing radiation. Cancer Res., 51: 4565â€”4569, transiently in BHK21 cells (16). It is well known that chromatin 1991. condensation is triggered by the phosphorylation of histone Hi (i7). 4. Hallahan, D. E., Virudachalam, S., Schwartz, J. L, Panje, N., Mustafi, R., and Weichselbaum, R. R. inhubutuonof protein kinases sensitizes human tumor cells to Furthermore, the inhibition of phosphatases 1 and 2A phosphorylates ionizing radiation. Radiat. Res., 129: 345â€”350,1992. two chromatin-associated proteins, pp33 and histone H3, which may 5. Kato,Y., Fusetani,N.,Matsunaga,S.,Hashimoto,K.,Fujita,S., andFuruya,T.J. Calyculin A, a novel antitumor metabolite from the marine sponge Discodermia represent an early nuclear signal for the modulation of nucleosomal calyx. J. Am. Chem. Soc., 108: 2780â€”2781,1986. structure (18). Histone Hi and H3 are phosphorylated in tsBN2 cells 6. Tosuji, H., Mabuchi,I., Fusetani,N., and Nakazawa,T. CalyculinA induces con tractile ring-like apparatus formation and condensation of chromosomes in unfertil incubated at the nonpermissive temperature (19). We postulate that ized sea urchin eggs. Proc. Nati. Acad. Sci. USA, 89: 10613-10617, 1992. such proteins which are associated with chromatin conformation are 7. Dewey, W. C., Noel, J. S., and Dettor, C. M. Changes in radiosensitivity and the key proteins in the enhancement of X-ray cell killing by CL-A. dispersion of chromatin during the cell cycle of synchronous Chinese hamster cells. Radiat. Res., 52: 373â€”394,1972. Recently, there has been strong evidence for the involvement of 8. Dettor, C. M., Dewey, W. C., Winans, L F., and Noel, J. S. Enhancement of X-ray protein kinases in the radiation response of cells. Woloschak et a!. (2) damagein synchronousChinesehamstercells by hypertonictreatments.RadialRes., 52:352â€”372,1972. have reported that X-irradiation induces the expression of protein 9. Pantelias,G. E. Radiation-inducedcytogeneticdamagein relationto changesin kinase C specific mRNA within 1 h. A 5-fold increase in protein interphase chromosome conformation. Radiat. Res., 105: 341â€”350,1986. 10. Sasaki, H., and Nishimoto, T. X-ray-related potentially lethal damage expressed by kinase C activity occurs in HL-60 cells exposed to ionizing radiation chromosome condensation and the influence of caffeine. Radiat. Res., 120: 72â€”82, (3). The weel protein kinase is required for mitotic block following 1989. radiation exposure in fission yeast (1). The inhibition of protein ii. Sasaki, H., and Nishimoto, T. Chromosome condensation may enhance X-ray-related celllethalityina temperature-sensitivemutant(tsBN2)ofbabyhamsterkidneycells kinases increases radiosensitivity in human tumor cell lines (4). These (BHK21). Radiat. Res., 109: 407â€”418,1987. findings indicate an important role for protein kinase-mediated signal 12. Chartier, L, Rankin, L L, Allen, R. E., Kato, Y., Fusetani, N., Karaki, H., Watanabe, S., andHartshorne,D.3.CalyculinAincreasesthelevelof proteinphosphorylation transduction in repair processes of X-irradiated cells. However, pro and changes the shape of 3T3 flbroblasts. Cell Motil. Cytoskeleton, 18: 26â€”40,1991. tein phosphorylation level is also negatively regulated by protein 13. Reddy, N. M. S., and Lange, C. S. Serum, trypsin, and cell shape but not cell-to-cell contact influence the X-ray sensitivity of Chinese hamster V79 cells in monolayers phosphatases. Considering the fact that the inhibition of phosphatases andin spheroids.Radiat.Res.,127:30â€”35,1991. increases phosphorylation of proteins (12, 15), it is reasonable to 14. Schneiderman, M. H., Dewey, W. C., Leeper, D. B., and Nagasawa, H. Use of the suppose that CL-A interferes with the radiation response through niitotic selection procedure for cell cycle analysis. Exp. Cell Res., 74: 430â€”438, 1972. â€œunbalancedâ€•signalingin which hyperphosphorylation stimulates. 15. Haystead, T. A., Sim, A. T., Caning, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and We postulate that CL-A-induced radioenhancement is partly associ Hardie, D. G. Effects of the tumor promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature (Land.), 337: 78â€”81,1989. ated with this â€œunbalancedâ€•signalingevent during repair processes, 16. Yamashita, K., Yasuda, H., Pines, J., Yasumoto, K., Nushitani, H., Ohtsubo, M., although there is no direct evidence to support this hypothesis. Hunter, T., Sugimura, T., and Nishimoto, T. Okadaic acid, a potent inhibitor of type 1 andtype 2A proteinphosphatases,activatescdc2/H1 kinaseandtransientlyinduces Our data presented here show that the radiosensitivity of BHK21 a prematuremitosis-likestate in BHK21 cells. EMBOJ., 9: 4331-4338, 1990. cells is markedly enhanced by CL-A treatment. Protein phosphatases 17. Bradbury, E. M., highs, R. J., and Matthews, H. R. Control of cell division by very may represent a new strategic target in the combined treatment of lysine rich histone(Fl) phosphorylation.Nature(Land.), 247: 257â€”261,1974. 18. Mahadevan, L. C., Willis, A. C., and Barratt, M. J. Rapid histone H3 phosphorylation cancer with radiation. Okadaic acid, which is structurally distinct from in response to growth factors, phorbol esters, okadaic acid, and protein synthesis CL-A, also induces chromatin condensation in BHK21 cells (i6). inhibitors. Cell, 65: 775-783, 1991. 19. Ajiro, K., Nishimoto, T., and Takahashi,T. Histone Hi and H3 phosphorylation However, okadaic acid was 100-fold less effective in enhancing X-ray during premature chromosome condensation in a temperature-sensitive mutant cell killing in BHK21 cells.3 Thus, we believe that CL-A, or more (tsBN2) of baby hamster kidney cells. J. Biol. Chem., 258: 4534â€”4538,1983. potent derivatives, will be clinically useful in radiotherapy. Further studies need to be carried out in animal model systems. 3 K. Nakamura and S. Antoku, unpublished observation.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. Enhancement of X-Ray Cell Killing in Cultured Mammalian Cells by the Protein Phosphatase Inhibitor Calyculin A

Katsumasa Nakamura and Shigetoshi Antoku

Cancer Res 1994;54:2088-2090.

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