Int. J. Cancer: 50,438-442 (1992) 0 1992 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer

INCREASED PHOSPHORYLATION OF NUCLEAR PHOSPHOPROTEINS IN HUMAN LUNG-CANCER CELLS RESISTANT TO cis-DIAMMINEDICHLOROPLATINUMf 11) Kazuto NISHIO', Yoshikazu SUGIMOTO', Kazuo KASAHAFU',Yasuhiro FUJIWARA', Shinji NISHIWAK?, Hirota FUJIKI~, Masahiro OHATA3 and Nagahiro SAIJ0'.4 'PharmacologyDivision and 'Cancer Prevention Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104; and 'Department of Internal Medicine, Kihoku-Hospital, Wakayama Medical College, Myoji 21 9, Katsuragi-cho, Ito-gum, Wakayama 649-71, Japan. A novel non-phorbol-ester-liketumor promoter, okadaic acid (OA) has been shown to be an inhibitor of protein phosphatase I and IIA and, thus, to cause an "apparent activation" of protein kinase C (PKC). We previouslyshowed that cis-diamminedichloroplatinum(l1) (CDDP)-resistant cells, PC-9/CDDP, were crossresistant to OA and that the cross-resistance was not due to the increased efflux of OA. We hypothesizedthat the phosphorylation status of some cellular proteins might be important in CDDP-resistance. No significant difference in PKC activity or total protein phosphatase activity measured in vitro was seen between PC-9 and PC-9/CDDP cells, nor in their sensitivity to inhibition by OA, nor in the amount of phosphorylationof whole cells or TCA-insoluble material. By SDS-PAGE after incubation of intact cells with 12P,we detected a marked increase, compared to PC-9 cells, in phosphorylation of the nuclear proteins of MW 32 and 20 kDa in CDDP-resistant PC-9/CDDP cells with no apparent difference in protein content. When phosphorylation of nuclear proteins observed in PC-9/CDDP cells was analyzed by 2-dimensional SDS-PAGE, the 32-kDa protein had a PI of about 4.5. The 32-kDa and 20-kDa bands were increased in a dose-dependent manner by CDDP treatment. On the other hand, no increase in phosphorylation of these proteins was observed in parental PC-9 cells. These results demonstrate a marked difference in the phosphorylation status of specific nuclear proteins between parental and CDDP-resistant cell lines, which may be related to CDDP-resistance.

phosphate-buffered saline (PBS) were purchased from Nissui (Tokyo). Fetal bovine serum (FBS) was purchased from Bocknek (Toronto, Canada). ['2P]orthophosphate and [3H]phorbol12, 13-dibutyrate (['H]PBt2) were purchased from Amersham Japan (Tokyo). Okadaic acid were isolated from a black sponge, Halichondria okadai, collected from the coast of Mie Prefecture as reported (Suganuma et al., 1988). 12-0-tetradecanoylphorbol 13-acetate (TPA) and okadaic acid were dissolved in 10% DMSO (Wako, Osaka, Japan) and stored at -70°C until use. Other drugs and chemicals were purchased from Sigma (St. Louis, MO) if not otherwise mentioned.

cis-diamminedichloroplatinum (11) (CDDP) is one of the most potent anti-cancer agents. To elucidate the mechanism of CDDP-resistance, we have established CDDP-resistant sublines (PC-9ICDDP) in vitro from a non-small-cell lung-cancer cell line, PC-9. Mechanisms for CDDP-resistance are multifactorial (Bungo et al., 1990; Fujiwara ef al., 1990). The main mechanism has not yet been determined; however, CDDPresistance appears to be quite different from the multi-drugresistance phenotype. We have reported that a CDDPresistant human lung-cancer cell line was cross-resistant to the growth-inhibitory effect of okadaic acid (Nishio et al., 1990). We speculate that certain mechanisms are common to CDDPresistance and okadaic-acid-resistance and that elucidation of the biochemical action of okadaic acid in the CDDP-resistant cells may give some clue to identification of a new CDDPresistant mechanism. The liberation of phosphate from phosphorylated histone type-111-S after incubation with protein phosphatases I and IIA is inhibited by okadaic acid (Suganuma et al., 1988).This inhibition results in the "apparent activation" of protein kinases (Sassa et ul., 1990). The effects of okadaic acid appear to be largely mediated through signal transduction pathways involving protein phosphatase inhibition. In the present study, we examined the phosphorylation of cellular components, involving the activities of PKC and protein phosphatases, and analyzed these substrates in CDDPresistant cells as compared with parental cells.

Assay of phosphorylation in whole cells and in TCA-insoluble materials Cells (1 x lo5 cells/ml) were placed in 10-cm tissue culture dishes, containing 10 ml of RPMI 1640 medium supplemented with 10% FBS. After incubation for 24 hr, ["'P]orthophosphate was added to a final concentration of 0.5 mCi/ml and the cell suspensions were incubated for another 2% hr. To determine the phosphorylation level in whole cells, the labelled cells were harvested and washed with cold PBS 3 times. Cells were collected by centrifugation (600g for 5 min). To determine the phosphorylation level in TCA-insoluble materials, labelled cells were harvested and washed 3 times with cold PBS. Cells were collected in a centrifugation tube by centrifugation (600g for 5 min) and ice-cold 10% TCA was added. After standing on ice for 15 min, the samples were centrifuged at 10,000g for 15 min at 4°C. The supernatants were decanted and washed with


Chemicals CDDP was obtained from Bristol-Myers Squibb (Tokyo, Japan). RPMI 1640 and Ca2+-freeand Mg2+-freeDulbecco's

Cell lines The human lung-cancer cell line, PC-9, derived from a previously untreated patient with adenocarcinoma of the lung, was provided by Prof. Y. Hayata, Tokyo Medical College. Its CDDP-resistant subline, PC-9/CDDP, was established by exposure of PC-9 to stepwise increasing CDDP concentrations and was selected by limiting dilution. This cell line showed 4.2-fold resistance to CDDP compared to its parental cell line PC-9, based on the 50% inhibitory concentrations (IC5,,) in 3-[4,5 dimethylthiazol 2-yl], 2,5-diphenyltetrazolium bromide (MTT') assay with continuous drug exposure (Hong et al., 1988).

4To whom correspondence and reprint requests should be addressed. Abbreviations:CDDP, cis-diamminedichloroplatinum(I1);PKC, protein kinase C; PBS, calcium-free and magnesium-free Dulbecco's phosphate-buffered saline; FBS, fetal bovine serum; PBt,, phorbol dibutylate; DTT, dithiothreitol: TPA, 12-0-tetradecanoylphorbol 13acetate; DMSO, dimethylsulfoxide; MTT, 3-(43 dimethylthiazol2-yl], 2,5-diphenyltetrazolium bromide; TCA, trichloroacetic acid; EDTA, ethylenediamine tetraacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; 2-D PAGE, 2-dimensional polyacrylamide gel electrophoresis; MW, molecular weight; kDa, kilodalton; OA, okadaic acid; PI, isoelectric point. Received: June 11,1991 and in revised form September 20,1991.




SDS-PAGE analysis ofphosphotylation of nuclearproteins and whole cells Cells (2 x lo5)were placed in 10-cm tissue culture dishes, containing 10 ml of RPMI 1640 medium supplemented with 10% FBS. After incubation for 24 hr, [3ZP]orthophosphatewas added to a final concentration of 0.5 mCi/ml and incubation was continued for another 3 hr. The cell lysate was prepared [3H]PBt,binding to PC-9 cells according to a modification of the method of Richter et al. Cells (1 x lo6cells/ml) were incubated for 30 rnin at 37°C in (1983). The cell suspensions were centrifuged at 600 g for 5 medium containing 20 nM [3H]PBt, (specific activity; 17.6 min, and decanted. Then the cells were washed twice with cold Ci/nmol). Cell suspensions were exposed to various concentra- PBS, harvested and lysed in ADOC buffer consisting of 1% tions of okadaic acid or TPA for 2 hr. After exposure to Nonidet P-40 (Iwai, Tokyo), 7 mM NaCl, 7 mM Tris-HC1, and 1 CDDP, all samples were filtered onto 0.65 pm (pore size) mM MgCl,, pH 7.4. Samples were concentrated by acetone Durapore membranes (Nihon Millipore Kogyo, Yonezawa, precipitation for 12 hr at -70°C as whole-cell extracts. The Japan). The membranes were washed 3 times with PBS and nuclei were isolated by centrifugation at 600 g for 10 rnin and the radioactivities were measured in a Beckman (Tokyo) the supernatant was decanted, then the nuclear pellets were LS3801 liquid scintillation counter. The results are expressed dissolved in SDS sample buffer. For protein analysis by in terms of specific PBt, binding, which is the difference one-dimensional SDS-PAGE, phosphorylated proteins in the between bindings in the absence and the presence of 10 p~ nuclear fraction or whole cells were analyzed after electrounlabelled PBt,. All experiments were performed in duplicate phoresis (Laemmli et al., 1970) in a 10% or 10-20% gradient and the data are presented as the means of at least 3 SDS polyacrylamide gel (84 x 90 x 1.0 mm) (Daiichi, Tokyo). independent experiments. Prior to gel electrophoresis, all samples were equalized for radioactivity and denatured at 100°Cfor 10 min. The gels were fixed with 20% methanol/l5% acetic acid for 1 hr, and stained Effect of protein phosphatase activity Cells in logarithmic phase of growth (5 x lo6) were har- with Coomassie brilliant blue (Daiichi), then dried with a vested 4 days after passage, then washed with ice-cold PBS. Model 583 gel dryer (Bio-Rad, Tokyo). Labelled proteins were The cells were homogenized with 50 mM Tris-HC1, pH 7.4, 2 detected by exposing Kodak hyperfilm MP (Kodak, Tokyo) to mM 2-mercaptoethanol, 0.25 M sucrose, 2 mM EDTA, 2 mM the dried gels. Low-molecular-weight markers (Bio-Rad) were ethylene glycol bis( p-aminoethyl ether)-N,N,N',N'-tetraacetic used for standards. acid, 10% glycerol, 0.2 mM leupeptin, 0.2 nM phenylmethylsulfonyl fluoride, and sonicated at 800 J with a Branson Model 2D-PAGE analysis of nuclearphosphoproteins Nuclear extracts were prepared as described above. The 450 sonifier (Branson, Danbury, CT). The homogenate was centrifuged at 1,OOOg for 30 rnin and the resulting supernatant nuclear pellets were dissolved in 8 M urea, 2% Nonidet P-40, was centrifuged at 10,000 g for 60 rnin at 10°C. The resulting 5% 2-mercaptoethanol, 2% Ampholinem carrier ampholyte, supernatant was diluted to various concentrations of protein PH 4.0-6.5 (Pharmacia, Uppsala, Sweden). Two-dimensional and used to assay protein phosphatase activity as described PAGE was carried out in the first dimension on Ampholinea ) 1 polyacrylamide gel plate (PH 4-6.5) (Pharmacia) by the modi(Ingebritsen and Cohen, 1983). Briefly, a sample (25 ~ 1 with pg of ["PI histone H, was incubated for 10 rnin at 30°C in the fied method of O'Farrell et al. (1975, 1977). The second final volume of the assay mixture (0.05 M Tris-HC1, pH 7.0, 1 dimension electrophoresiswas carried out in 10-10% gradient gels mM EDTA, 1% 2-mercaptoethanol, 0.003% Brij 35 (w/v), 0.3 in the presence of SDS. Gels were fixed and dried as described mg/ml BSA, 2 mM MnC1,). The reaction was terminated by previously and exposed on Kodak hyperfilm MP for 48 hr. addition of 2 ml of 2.5% TCA, and filtered. The filter paper was dried and the radioactivity counted. Mouse brain protein Effect of okadaic acid or CDDP on the 32-kDa and 20-kDa phosphatase was used as a control. Protein phosphatase nuclearphosphoproteins in PC-9and PC-9ICDDP cells PC-9 and PC-9/CDDP cells (2 x 105/ml) were placed in activity was expressed as the remaining ["PI in phosphorylated 10-cm tissue culture dishes, containing 10 ml of RPMI 1640 histone H, in the presence of okadaic acid.

10 vol of ethanol/ether (Wako) (1:l) (vol/vol) with brief mixing. After centrifugation at 10,000g for 15 rnin at 4"C, the supernatants were decanted and dried under N, gas. The protein content was determined by a modification of the technique of Lowry et al. (1951).



5000 1


- 1

!= .al 5

loool 04 0




E, 5? I



Time (min)




04 0




Time (min)

FIGURE 1- Effect of okadaic acid ( u ) and TPA (b) on protein kinase C activity in PC-9 (-0-)and PC-9/CDDP (-*--) Protein kinase C activity was assayed by [3H]PBt,binding.




medium supplemented with 10% FBS. For the time-course study of okadaic acid treatment, 200 ng/ml of okadaic acid were added 0,15,30,60 and 120 min before termination of the reaction. To study the dose-response of CDDP, cells were treated with various concentrations of CDDP for 12 hr. After incubation, [32P]orthophosphatewas added to a final concentration of 0.5 mCi/ml and incubation was continued for another 3 hr. Nuclear extracts were prepared and SDS-PAGE analysis was performed as described above. RESULTS

Okadaic acid has been shown to cause an “apparent activation” of PKC. Therefore, we examined the effect of okadaic acid on PKC activity by [3H]PBt2binding assay. There was no difference PKC activity between the untreated PC-9 and PC-9/CDDP cells, while okadaic-acid treatment slowly increased PKC activity in a time-dependent manner (Fig. la). Figure lb shows the effect of TPA on the 2 cell types. TPA activates PKC activity transiently and only slightly in the early phase. However, there was no difference in response between the 2 cell types. We also checked the effect of CDDP on PKC activity. In our experiments, short-term treatment of CDDP had no effect on PKC activity (data not shown).



80 60 40 -

Okadaic acid is a protein-phosphatase inhibitor, as described previously. Therefore, we also checked the proteinphosphatase activities in PC-9 and PC-9/CDDP cells. Proteinphosphatase activity was measured as the release of phosphate from phosphorylated histone HI. There was no difference in protein-phosphatase activity between untreated PC-9 (IDso= 3.79 2 0.81 kM/mg protein/min) and PC-9/CDDP (IDso= 4.10 h 0.94 pM/mg protein/min) cells. Okadaic acid inhibited protein-phosphatase activities in both PC-9 and PC-9/CDDP cells to the same degree (Fig. 2). This suggested that protein-phosphatase activity, inhibited by okadaic acid, was not responsible for cross-resistance to CDDP and okadaic acid. To confirm these results, the growth-inhibiting effect of sodium fluoride, which acts as a non-specific proteinphosphatase inhibitor, was examined in drug-sensitive and -resistant cells by MTT assay. There was no significant difference in the IC,, values of sodium fluoride between PC-9 (1.8 kg/ml) and PC-9/CDDP (2.4 pg/ml) (data not shown), and sodium fluoride did not show any sensitizing effect of CDDP on cells of both types (data not shown). We examined the amount of incorporation of [”Plorthophosphate into whole cells and TCA-insoluble materials after treatment with okadaic acid. There was no significant difference in the amount of phosphorylation of whole cells or TCA-insoluble fractions between PC-9 and PC-9ICDDP (Fig. 3). We therefore concluded that CDDP-resistance in PC-91 CDDP cells was not due to the difference in the amount of incorporation of [32P]orthophosphateinto whole cells. Then we analyzed the substrate-specificity of phosphorylation or dephosphorylation. Phosphorylation of nuclear proteins was examined by SDSPAGE after incubation of intact cells with [32P]orthophosphate. Reproducible changes occurred in a few nuclear phosphoproteins of PC-9/CDDP. An increase was observed in the phosphorylation of the nuclear phosphoproteins, consistent with molecular weights (MW) of 32 and 20 kDa (Fig. 4u). The

20 E


0‘ 10”





okadaic acid (pM)


FIGURE 2 - Inhibition of protein phosphatase activity by okadaic acid in PC-9 and PC-9/CDDP cells. The protein phosphatase activity was measured as the radioactivity of the remaining [32P]orthophosphatein phosphorylated histone H, incubated with cell extracts of PC-9 (-0-), PC-9/CDDP (-0-) cells.


-?3 z


whole cells




43 E


1 r m


r 0 Q


TCA insoluble fraction

U ’







Time (h)

FIGURE 3 - Effect of okadaic acid on the amount of incorporation of (32P]orthophosphateinto whole cells [PC-9 (-0-), PC-9ICDDP (-0-) cells] and TCA-insoluble materials [PC-9 (-El-), and PC-9ICDDP (--.--)I.

FIGURE 4 - ( a ) Autoradiographs of the phosphorylated nuclear proteins from PC-9 and PC-g/CDDP cells. Growing cells were Incubated with [32P]orthophosphatefor 2% hr. Nuclei were isolated and the nuclear extracts were adjusted to 2 x lo6 cpm and analyzed by SDS gel electrophoresis followed by autoradiography. ( b ) Coomassie blue staining of nuclear proteins from PC-9 and PC-9/CDDP cells.



FIGURE 5 - Autoradiograghsfrom 2-D PAGE of phosphorylated nuclear proteins of PC-9 cells and PC-g/CDDP cells after incubation in the presence of 0.5 mCi [ Plorthophosphate for 3 hr. Autoradiographswere exposed for 48 hr.

32-kDa protein was not affected by okadaic acid (Fig. 6). On the other hand, okadaic acid time-dependently enhanced the phosphorylation status of the phosphoprotein with an MW of about 20 kDa. This suggests that the 20-kDa phosphoprotein is a substrate of the protein phosphatase inhibited by okadaic acid. Although the phosphorylation of 20-kDa protein was also observed in PC-9 cells, it was detected later than that which occurred in PC-9/CDDP cells (60 min us. 15 min: PC-9 vs. PC-9/CDDP). When PC-9/CDDP cells were treated with several concentration of cisplatin for 12 hr, the phosphorylation of both the 32-kDa (Fig. 7 a ) and the 20-kDa proteins (Fig. 7b) was increased, but not in PC-9 cells. DISCUSSION

We previously showed that CDDP-resistant cells, PC-9/ CDDP, were cross-resistant to okadaic acid. Okadaic acid is an inhibitor of protein phosphatase and thus causes an “apparent activation” of PKC. Therefore, we hypothesized that the phosphorylation status might be important in CDDP-resistance. However, several studies have shown that TPA modulates FIGURE6-Time course of the effect of okadaic acid on CDDP-induced cytotoxicity (Hofmann et al., 1988; Basu and phosphorylation of the nuclear protein in PC-9 and PC-9/CDDP Lazo, 1990; Isonishi et al., 1990). Hofmann et al. observed cells. Cells were isolated and pre-labelled with [3ZP]orthophos- sensitization of Walker rat carcinoma cells to CDDP by phate for 30 min before the addition of okadaic acid. Okadaic acid long-term (48 hr) exposure to TPA and postulated that the (200 p,g/ml) was added at various time points. Cells were collected sensitization effect of TPA resulted from the inhibition or and homogenized, and a nuclear fraction was prepared as de- down-regulation of PKC. Basu and Lazo (1990) also observed scribed in “Material and Methods.” Samples of the nuclear that long-term (24 hr) pre-treatment with TPA sensitized fraction were mixed with an equal volume of sample buffer, then HeLa cells to CDDP, but showed that down-regulation of the sonicated and denatured. All samples were adjusted to 2 x lo4 cpm/ml, and applied to electrophoresis in a 10-20% gradient PKC could not explain the sensitizing effect of TPA and polyacrylamide gel. The gels were fixed, dried and autoradio- postulated that activation of PKC was necessary for sensitization to CDDP. Isonishietal. (1990) reported that short-term (1 graphed at -70°C for 48 hr. hr) TPA exposure could sensitize 2008 ovarian carcinoma cells to CDDP. Although they did not measure the actual PKC phosphoprotein spots that underwent changes in phosphate activity, they suggested that CDDP sensitivity could be moducontent corresponded to minor spots on the Coomassie-blue- lated by PKC. However, the role of PKC in the modulation of stained gel (Fig. 4b) or silver staining (data not shown). When CDDP-induced cytotoxicity was not clear. we analyzed the nuclear phosphoprotein by 2D-PAGE, the In this report, we show that there is no significant difference 32-kDa band, which is consistent with the former phosphopro- between CDDP-resistant cells and their parental cells in the tein seen in one-dimensional SDS-PAGE, appeared in PC-9/ activities of PKC and protein phosphatase and the incorporaCDDP cells but not in PC-9 cells (Fig. 5). The 32-kDa band has tion of [32P]orthophosphate into whole cells and TCAa PI of 4.5. On 2-dimensional analysis using PIE with 4-6 PH insoluble materials. Cells of both types showed the same range as the first dimension, we could not identify the 20 kDa response to treatment with okadaic acid and TPA. It appears phosphoprotein which appeared in one-dimensional SDS- from these results that regulation of the amount of phosphoryPAGE. lation by kinases or phosphatases is not responsible for In PC-9/CDDP, okadaic acid (200 ngiml) induced phospho- CDDP-resistance. Therefore, we analyzed the substrate of rylation of some nuclear proteins in a time-dependent manner phosphorylation or dephosphorylation. Two nuclear phos(Fig. 6). However, the amount of phosphorylation of the phoproteins with MW of 32 kDa (PI 4.5) and 20 kDa were



FIGURE 7 - Dose-response of the effect of CDDP on phosphorylation of the nuclear protein in PC-9 and PC-9/CDDP cells. Cells were treated with various concentrations of CDDP for 12 hr, then collected and homogenized, after which a nuclear fraction was prepared as described in “Material and Methods”. Samples of the nuclear fraction were mixed with an equal volume of sample buffer, then sonicated and denatured. All samples were adjusted to 2 X lo4 cpmiml, and applied to electrophoresis in 10% ( a ) or 10-20% ( b ) gradient polyacrylamide gel. The gels were fixed, dried and autoradiographed at -70°C for 48 hr. specifically observed only in PC-9/CDDP cells. T h e phosphorylation of these 2 nuclear phosphoproteins was increased by CDDP treatment. These results suggested that these nuclear phosphoproteins may be related to CDDP-resistance, although their functions were not clear. One possibility is that the nuclear phosphoproteins may act o n DNA damage, which is t h e final target of C D D P (Eastman, 1987). We are therefore analyzing the function of these proteins on D N A damage in CDDP-resistant cells. It was not clear whether the phosphorylation change was d u e t o the change in t h e level of phosphorylation or in the level of protein content. T h e various activities

of many nuclear phosphoproteins, such as topoisomerases, were regulated by phosphorylation and dephosphorylation (Pommier et al., 1990; Gorsky e t al., 1989). Thus, w e speculate that t h e phosphorylation of these phosphoproteins seen in CDDP-resistant cells is regulated by signal transduction pathways involving phosphorylation. ACKNOWLEDGEMENTS

K.N. was the recipient of a research resident fellowship from the Foundation for Promotion of Cancer Research.


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Increased phosphorylation of nuclear phosphoproteins in human lung-cancer cells resistant to cis-diamminedichloroplatinum (II).

A novel non-phorbol-ester-like tumor promoter, okadaic acid (OA) has been shown to be an inhibitor of protein phosphatase I and IIA and, thus, to caus...
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