]CANCER RESEARCH 53,239-241, January 15, 19931

Advances in Brief

Hyperphosphorylation of Retinoblastoma Protein and p53 by Okadaic Acid, a Tumor Promoter

Jun Yatsunami, Atsumasa Komori, Tetsuya Ohta, Masami Suganuma, and Hirota Fujiki 2 Cancer Prevention Division, National Cancer Center Research Institute, Tokyo 104, Japan

Abstract

A potent tumor promoter, okadaic acid, induced hyperphosphorylation of tumor suppressor proteins, retinoblastoma protein and p53, by in vitro

incubation with nuclei isolated from rat regenerating liver as well as by incubation with primary human fibroblasts. Most of the retinoblastoma protein migrated to a hyperphosphorylated position in electrophoresis. The phosphorylation of p53 was increased at a rate 8 times that in non- treated primary human fibroblasts. Hyperphosphorylation of tumor sup- pressor proteins, mediated through inhibition of protein phosphatases 1 and 2A, is involved in tumor promotion by okadaic acid. The significance of hyperphosphorylation of the retinoblastoma protein and p53 is dis- cussed in relation to the regulation of the cell cycle.

Introduction

Okadaic acid class tumor promoters, which are potent inhibitors of

protein phosphatases 1 and 2A, induce tumors from initiated cells in various organs, mediated through common biochemical and molecular

processes (1). Their mechanisms of action are named the okadaic acid

pathway; i.e., okadaic acid binds to the okadaic acid receptors, cata-

lytic subunits of protein phosphatases 1 and 2A; inhibits their activ-

ities, resulting in increased phosphorylation of cellular and nuclear

proteins; and induces expression of genes responsible for cell prolif- eration.

The tumor suppressor gene products, Rb 3 protein and p53, which are nuclear phosphoproteins, are proposed to regulate cell growth in a

negative manner (2). Although the loss or inactivation of tumor sup-

pressor genes in human and rodent tumors has been extensively an-

alyzed (3, 4), alteration of the tumor suppressor genes in mouse skin

two-stage carcinogenesis was not often found (5). If the alteration in

the tumor suppressor genes is a later event in the process of tumor

promotion and tumor progression, how can tumor suppressor gene

products be involved in a process of tumor promotion? On the basis

of evidence that hyperphosphorylated forms of Rb protein and p53 are

inactive forms, we recently hypothesized that sustained hyperphos-

phorylation of these proteins is an alternative explanation for the loss

of function of both tumor suppressor gene products in the okadaic acid pathway (6).

To identify hyperphosphorylat ion of the Rb protein and p53, nuclei

isolated from rat regenerating liver and primary human fibroblasts were treated with okadaic acid. The tumor suppressor proteins were

precipitated with specific monoclonal antibodies. Hyperphosphoryla- tion of the Rb protein was estimated by incorporation of 32P i into Rb

protein for in vitro incubation with the nuclei and by the migration to

Received 11/2/92; accepted 11/30/92. 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 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture; a grant from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control, Japan; and grants from the Foundation for Promotion of Cancer Research, the Uehara Memorial Life Science Foundation, and the Princess Takamatsu Cancer Research Fund.

2 To whom requests for reprints should be addressed. 3 The abbreviations used are: Rb, retinoblastoma; TPA, 12-O-tetradecanoylphorbol-

13-acetate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

a hyperphosphorylated Rb position in one-dimensional SDS-PAGE for primary human fibroblasts labeled by [35S]methionine. The hy-

perphosphorylation of p53 was autoradiographically determined for both cases. These findings are the first to describe the hyperphospho-

rylation of tumor suppressor gene products induced by a tumor pro-

moter, okadaic acid.

Materials and Methods

Okadaic acid was isolated from the black sponge, Halichondria okadai (7). [3~-32p]ATP, [35S]methionine, and 32pi were obtained from Amersham, Buck- inghamshire, United Kingdom. Male Fischer 344 rats, 7 weeks old, were purchased from Charles River Japan, Inc., Kanagawa, Japan. The nuclei (200 lag), which were isolated from the rat liver 2 days after partial hepatectomy and from the liver without hepatectomy (8), were incubated with 2.5 ]aM ['y-32p]ATP (3.7 MBq/ml) and various concentrations of okadaic acid in a buffer containing 50 rnM Tris-HC1 (pH 7.6), 10 n ~ MgC12, 1 mM dithiothreitol, and 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid for 10 min at 30~ After re- action, the nuclei were immediately solubilized in 1 ml of the lysis buffer containing 25 mM Tris-HCI (pH 7.4), 50 rnM NaCI, 0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, and 50 pg/ml aprotinin and then centrifuged at 12,000 • g for 10 min. The supernatants were treated with either an anti-Rb protein antibody (G3-245; PharMingen) or an anti-p53 antibody (PAb421; Oncogene Science) in ice- water for 2 h. Each immunocomplex was absorbed on protein A-Sepharose 4B (Pharmacia) in ice-water for 1 h and washed 3 times with the lysis buffer. Immunoprecipitates were subjected to SDS-PAGE, and the radioactivity was determined by BAS-2000 Image Analyzer (Fuji Film Co., Tokyo, Japan). Their radioactivities in nuclei isolated from regenerating and normal livers were determined by five and three different experiments, respectively.

Primary human fibroblasts (5 • 105 cells) were placed in a culture dish with 4 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (9). For the study of Rb protein phosphorylation in the cells, Rb protein was labeled with [35S]methionine, rather than 32p~. Since hyperphos- phorylated Rb protein has a unique feature which causes it to migrate differ- ently than the hypophosphorylated Rb protein in SDS-PAGE, this was taken as a parameter to estimate the degree of Rb protein phosphorylation. The medium was replaced by a methionine-deficient medium and incubated for 14 h. [35S]- Methionine, at a concentration of 3.7 MBq/ml, was added to the medium and it was further incubated for 3 h. The cells were treated with okadaic acid at various concentrations for length of times indicated in the text. The cells were solubilized in the lysis buffer. The supematants were immunoprecipitated with the anti-Rb protein antibody, and the immunoprecipitates were subjected to 7% SDS-PAGE. Radioactivity was determined by BAS-2000 Image Analyzer. As for p53 phosphorylation, the cells were incubated with phosphate-deficient Dulbecco's modified Eagle's medium for 14 h and labeled with 32Pi at a concentration of 7.4 MBq/ml for 3 h. The cells were treated with okadaic acid and solubilized, as described above. Cell lysates were immunoprecipitated with the anti-p53 antibody, the immunoprecipitates were subjected to 10% SDS- PAGE, and radioactivity was determined by BAS-2000 Image Analyzer.

Results and Discussion

Phosphorylat ion of Rb Protein and p53 in Nuclei in Vitro. In- cubation of the nuclei of the regenerating and normal liver with okadaic acid resulted in phosphorylation of numerous proteins, deter- mined by autoradiography of SDS-PAGE of the nuclear lysates (data

not shown). Immunoprecipi tat ion analysis with an anti-Rb protein

239

Research. on December 28, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

T U M O R S U P P R E S S O R P R O T E I N S A N D O K A D A I C A C I D

antibody revealed that the hypophosphorylated Rb protein has a mo-

lecular weight of 105,000 in SDS-PAGE and the hyperphosphorylated

Rb protein decreases its electrophoretic mobility, as reported previ-

ously (10, 11). Okadaic acid induced phosphorylation of Rb protein

linearly, at concentrations of 1 nM to 1 laM, by incubation with the

nuclei of the regenerating liver in vitro, whereas okadaic acid did not

increase the phosphorylation in the nuclei of normal liver. It is of

interest to note that the basal level of the phosphorylation in the nuclei

of the regenerating liver was twice as much as that in the nuclei of

normal liver (Fig. 1). The nuclei isolated from regenerating rat liver

are thought to contain activated protein kinases and protein phos-

phatases, whereas the nuclei isolated from normal liver are less active,

due to their remaining in Go. In regenerating liver, various protein

kinases, such as p34 cdc2 or cyclin-dependent kinase are activated,

because the Rb protein is phosphorylated by p34 cdc2 or its related

kinases (12). Carlberg et al. (13) reported that the highest protein

kinase activity was found in cells from the early S phase, whereas

protein phosphatase activity was most pronounced during G2 plus M.

These results are in agreement with our evidence that okadaic acid

was effective only on the nuclei of the regenerating liver, but not on

those of normal liver.

Phosphorylation of p53 in the nuclei isolated from regenerating

liver increased up to 100 nM okadaic acid and saturated at concentra-

tions of okadaic acid from 100 nM to 1 ~ (Fig. 2). However, phos-

phorylation of p53 in the nuclei isolated from normal liver with

okadaic acid was not increased significantly. In this case, the basal

levels of phosphorylation of p53 were the same with both kinds of

nuclei.

The difference between the dose-response curves of Rb protein and

p53 may be explained by various factors: (a) the half-life of Rb

protein is more than 10 h (11), whereas that of p53 is short, 5-20 min

(14); (b) Rb protein shows more than 20 tryptic phosphopeptides in

2-dimensional peptide mapping (15), whereas p53 shows 6 major

peptides (16); and (c) Rb protein has a molecular weight twice as large

._c O

o t). o3 to

o

,i.a

om "o

O .g.

tr

300

k 1 0 0 ,

0 I 1 I I 101 10 3

C o n c e n t r a t i o n ( n M )

Fig. 2. Phosphorylation of p53 protein in nuclei isolated from regenerating (0) and normal (Q)) rat liver by incubation with okadaic acid. Nuclei were isolated as described in Fig. 1. Nuclear lysates were immunoprecipitated with PAb421 and subjected to SDS- PAGE. Phosphorylation of p53 in nuclei of the normal liver was expressed as 100% and was the same as that of the regenerating liver. Phosphorylation of p53 was determined by the same procedure as that of Rb protein. Bars, SD.

1 2 3 4 5

105 kDa .--~

Fig. 3. Phosphorylation of Rb protein in primary human fibroblasts induced by okadaic acid. Cells labeled by [353]methionine were treated with okadaic acid at concentrations of 0, 1, 10, 100 and 1000 nM for 2 h (Lanes 1-5, respectively), kDa, molecular weight in thousands.

600

v e'-

-Q 4 0 0 n- ~6

o ='5 200

>

nt-

0 " - / / ' I I I i

101 10 3

C o n c e n t r a t i o n ( n M )

Fig, 1. Phosphorylation of Rb protein in nuclei isolated from regenerating (0) and normal (O) rat liver by incubation with okadaic acid. Nuclei isolated from the liver 2 days after partial 'hepatectomy and from normal liver were incubated with various concentra- tions of okadaic acid. Nuclear lysates were immunoprecipitated with G3-245 and sub- jected to SDS-PAGE. Radioactivity was analyzed by BAS-2000 Image Analyzer. Phos- phorylation of Rb protein in nuclei of the normal liver was expressed as 100% and that in nuclei of the regenerating liver was twice as much as that in the normal liver. Each point represents the mean _-_ SD (bars) of various experiments, as reported in "Materials and Methods."

as p53. In addition, the experimental results indicated that the Rb protein is a more stable phosphoprotein with more phosphorylation

sites than p53. Phosphory la t ion of Rb Prote in and p53 in P r i m a r y H u m a n

Fibroblasts. The hypophosphorylated and hyperphosphorylated Rb proteins, labeled by [35S]methionine, were differentiated by electro-

phoretic mobility. Both Rb proteins were well immunoprecipitated by an anti-Rb protein antibody, however. As Fig. 3, Lane 1, shows, primary human fibroblasts not treated with okadaic acid contained large amounts of hypophosphorylated Rb protein and small amounts of hyperphosphorylated Rb protein. Treatment with various concen- trations of okadaic acid for 2 h (Fig. 3, Lanes 2 to 5) decreased electrophoretic mobility of the Rb protein, depending on the concen- trations of okadaic acid, indicating that the Rb protein was hyper- phosphorylated in various degrees, and with 100 nM okadaic acid, most of the Rb protein was hyperphosphorylated. The time course of Rb protein phosphorylation in the cells treated with 100 nM okadaic acid was recorded for up to 240 min (data not shown). The Rb protein, which had started being phosphorylated within 30 min, shifted to hyperphosphorylated positions, depending on the incubation time (data not shown). These results were well correlated with the increase of phosphorylation of the Rb protein in the nuclei of the regenerating liver treated with okadaic acid in vitro.

The p53 was immunoprecipitated with an anti-p53 antibody from the lysates of the cells. Without okadaic acid treatment, p53 did not

240

Research. on December 28, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

TUMOR SUPPRESSOR PROTEINS AND OKADAIC ACID

show any strong phosphorylation. The phosphorylation of p53 was increased by treatment with okadaic acid, at concentrations of 100 nM and 1 pM, at a rate 8 and 9 times of that in nontreated primary human fibroblasts, respectively (Fig. 4A). Like the Rb protein, okadaic acid induced time-dependent phosphorylation of p53 in the cells that strongly increased at 60 rain (Fig. 4B). These results clearly indicated that phosphorylation of the Rb protein and p53 was induced through inhibition of protein phosphatases 1 and 2A by okadaic acid, i.e., the okadaic acid pathway.

This experiment clearly showed that a tumor promoter, okadaic acid, at concentrations of 1 nM to 1 iuM, induced phosphorylation of tumor suppressor gene products, probably resulting in inactivation of their function. We think that posttranslational modification is involved in the alteration of tumor suppressor function during tumor promotion. These concentrations were compared with those of okadaic acid, which was used for the 3-methylcholanthrene-induced transformation in BALB/3T3 cells (17). Two concentrations of okadaic acid, 12.5 and 25 nM, significantly induced cell transformation during 2-week treat- ment. Therefore, hyperphosphorylation of Rb protein and p53 is thought to be induced by okadaic acid during the process of cell transformation.

How are the hyperphosphorylated tumor suppressor proteins inac- tivated and how do they induce proliferation? It is reported that phosphorylated Rb protein loses contact with the cellular oncopro- teins, such as EIA, resulting in the removal of E2F from its complex (18). This allows the transcription factor to be activated. The conver- sion from hyperphosphorylated Rb protein to a hypophosporylated one during the M phase is induced by the presence of protein phos- phatase 1 (19), which is also inhibited by okadaic acid. Thus, okadaic acid might induce a cyclin-independent state and sustain the hyper- phosphorylated state, both of which cause deregulation of the cell cycle. It is also reported that p53 is phosphorylated at amino- and carboxyl-terminal sites and the amino-terminal sites are dephospho- rylated by protein phosphatase 2A (20). Therefore, the amino-terminal sites are hyperphosphorylated by okadaic acid. Although it is known

A 1 2 3 4 5

53 kDa,,,-~

that p53 causes a tetramer to become active (21), the molecules of hyperphosphorylated p53, which are unable to associate with each other, disturb the expression of downstream genes that negatively control growth (22). Our results first show that a tumor promoter, okadaic acid, induced hyperphosphorylation of two products of tumor suppressor genes at the same time. It has not yet been clarified whether phosphorylation of both proteins is involved in the process of tumor promotion or whether only one protein is actively involved. We simply assume that these hyperphosphorylated proteins might be sig- nificant factors regulating the proliferation of initiated cells.

B

53 kDa,--~

1 2 3 4 5

i , � 8 4

Fig. 4. Phosphorylation of p53 in primary human fibroblasts induced by okadaic acid. 20. (A) Cells labeled by 32p i for 3 h were treated with okadaic acid at concentrations of 0, 1, 10, 100 and 1000 nM, for 2 h (Lanes 1-5, respectively). (B) Cells were treated with 100 21. nM okadaic acid for 0, 15, 30, 60, and 120 min, respectively, p53 was immunoprecipitated with PAb421 and subjected to 10% SDS-PAGE. Radioactivity was analyzed by BAS-2000 22. Image Analyzer. kDa, molecular weight in thousands.

241

References

1. Fujiki, H., Suganuma, M., Nishiwaki, S., Yoshizawa, S., Yatsunami, J., Matsushima, R., Furuya, H., Okabe, S., Matsunaga, S., and Sugimura, T. Specific mechanistic aspects of animal tumor promoters: the okadaic acid pathway. In: R. D'Amato, T. J. Slaga, W. Farland, and C. Henry (eds.), Relevance of Animal Studies to the Evaluation of Human Cancer Risk, pp. 337-350. New York: John Wiley & Sons, Inc., 1992.

2. Weinberg, R. A. Tumor suppresser genes. Science (Washington DC), 254:1138-1146, 1991.

3. Benedict, W. E, Xu, H-J., Hu, S-X., and Takahashi, R. Role of the retinoblastoma gene in the initiation and progression of human cancer. J. Clin. Invest., 85: 988-993, 1990.

4. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. p53 Mutations in human cancers. Science (Washington DC), 253: 49-53, 1991.

5. Ruggeri, B., Caamano, J., Goodrow, T., DiRado, M., Bianch, A., Trono, D., Conti, C. J., and Klein-Szanto, A. J. P. Alteration of the p53 tumor suppressor gene during mouse skin tumor progression. Cancer Res., 51: 6615~621, 1991.

6. Fujiki, H. Is the inhibition of protein phosphatase 1 and 2A activities a general mechanism of tumor promotion in human cancer development? Mol. Carcinog., 5: 91-94, 1992.

7. Tachibana, K., Scheuer, E J., Tsukitani, Y., Kikuchi, H., Van Engen, D., Clardy, J., Gopichand, Y., and Schmitz, E J. Okadaic acid, a cytotoxic polyether from two marine sponges of the genus Halichondria. J. Am. Chem. Soc., 103: 2469-2471, 1981.

8. Hosoya, T., Nagai, Y., Inagaki, T., and Hayashi, M. In vivo effect of androgen and cycloheximide on the RNA synthesis in isolated nuclei of rat ventral prostates. J. Biochem. (Tokyo), 84: 151%1528, 1978.

9. Yatsunami, J., Fujiki, H., Suganuma, M., Yoshizawa, S., Eriksson, J. E., Olson, M. O. J., and Goldman, R. D. Vimentin is hyperphosphorylated in primary human fibro- blasts treated with okadaic acid. Biochem. Biophys. Res. Commun., 107:1165-1170, 1991.

10. DeCaprio, J. A., Ludlow, J. W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica-Worms, H., Huang, C-M., and Livingston, D. M. The product of the retinoblastoma suscep- tibility gene has properties of a cell cycle regulatory element. Cell, 58: 1085-1095, 1989.

11. Mihara, K., Cao, X-R., Yen, A., Chandler, S., Driscoll, B., Murphree, A. L., T'Ang, A., and Fung, Y. K-I. Cell cycle dependent regulation of phosphorylation of the human retinoblastoma gene product. Science (Washington DC), 246: 1300-1303, 1989.

12. Lin, B. T-Y., Gruenwald, S., Morla, A. O., Lee, W-H., and Wang, J. Y. J. Retinoblas- toma cancer suppressor gene product is substrate of the cell cycle regulator cdc2 kinase. EMBO J., 10: 858-864, 1991.

13. Carlberg, U., Nilsson, A., Skog, S., Palmquist, K., and Nygard, O. Increased activity of the eEF-2 specific, Ca 2+ and calmodulin dependent protein kinase III during the S-phase in Ehrlich ascites cells. Biochem. Biophys. Res. Commun., 180: 1372-1376, 1991.

14. Reich, N. C., Oren, M., and Levine, A. J. Two distinct mechanisms regulate the level of a cellular tumor antigen, p53. Mol. Cell. Biol. 3: 2143-2150, 1983.

15. Templeton, D. J., Park, S. H., Lanier, L., and Weinberg, R. A. Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering. Proc. Natl. Acad. Sci. USA, 88: 3033-3037, 1991.

16. Meek, D. W., and Eckhart, W. Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells. Mol. Cell. Biol., 8: 461-465, 1988.

17. Sakai, A., and Fujiki, H. Promotion of BALB/3T3 cell transformation by the okadaic acid class of tumor promoters, okadaic acid and dinophysistoxin-1. Jpn. J. Cancer Res., 82: 518-523, 1991.

18. Bagchi, S., Raychandhuri, E, and Nevins, J. R. Adenovirus E1A proteins can disas- sociate heteromeric complexes involve the E2F transcription factor: a novel mecha- nism for E1A trans-activation. Cell, 62: 659--669, 1990.

19. DeCaprio, J. A., Furukawa, Y., Ajchenbaum, E, Griffin, J. D., and Livingston, D. M. The retinoblastoma-susceptibility gene product becomes phosphorylated in multiple stages during cell cycle entry and progression. Proc. Natl. Acad. Sci. USA, 89: 1795-1798, 1992. Wang, Y., and Eckhart, W. Phosphorylation sites in the amino-terminal region of mouse p53. Proc. Natl. Acad. Sci. USA, 89: 4231-4235, 1992. Stenger, J. E., Mayr, G. A., Mann, K., and Tegtmeyer, E Formation of stable p53 homotetramers and multiples of tetramers. Mol. Carcinog., 5: 102-106, 1992. Vogelstein, B., and Kinzler, K. W. p53 Function and Dysfunction. Cell, 70: 523-526, 1992.

Research. on December 28, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from

1993;53:239-241. Cancer Res   Jun Yatsunami, Atsumasa Komori, Tetsuya Ohta, et al.   Okadaic Acid, a Tumor PromoterHyperphosphorylation of Retinoblastoma Protein and p53 by

  Updated version

  http://cancerres.aacrjournals.org/content/53/2/239

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/53/2/239To request permission to re-use all or part of this article, use this link

Research. on December 28, 2020. © 1993 American Association for Cancercancerres.aacrjournals.org Downloaded from