BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 242, 317–321 (1998)ARTICLE NO. RC977958

Cyclin E Overexpression Responsible for Growthof Human Hepatic Tumors with p21WAF1/CIP1/SDI1

Toshiya Tsuji,* Masahiro Miyazaki,* Kazuo Fushimi,* Koichiro Mihara,* Yusuke Inoue,*Ryuichiro Ohashi,* Motoaki Ohtsubo,† Keisuke Hamazaki,‡Shouji Furusako,§ and Masayoshi Namba*,1

*Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School,Okayama 700; †Division of Molecular Genetics, Institute of Life Science, Kurume University, Kurume 839;‡Center for Adult Diseases, Kurashiki 710; and §Mochida Pharmaceutical Co., Tokyo 115, Japan

Received December 2, 1997

which regulate the G1/S transition, are the major sub-We examined a relationship between p21WAF1/CIP1/SDI1

strates of the complexes of cyclin D/CDK4 and cyclinand cell-cycle-related proteins in 12 human liver tu- E/CDK2, respectively. Hypophosphorylated Rb proteinmor cell lines (JHH-1, -2, -4, -5, -6, -7; HLE; HuH-7; in G1 is active for growth suppression, while its phos-Hep3B; PLC/PRF/5; HuH-6; HepG2). Seven (JHH-1, -2,

phorylated counterpart in S/G2/M is inactive for-5, -6, -7; Hep3B; HepG2) out of eight cell lines havinggrowth suppression. p21WAF1/CIP1/SDI1, which can be in-p21WAF1/CIP1/SDI1 protein overexpressed cyclin E protein,duced by wild-type p53, inhibits the activity of thealthough one of them (JHH-5) overexpressed a re-cyclin D/CDK4, cyclin E/CDK2 and cyclin A/CDK2 com-duced size of cyclin E. The rest (HuH-6) of the 8 cellplexes, whereby the phosphorylation of Rb protein islines with p21WAF1/CIP1/SDI1 showed a decreased expres-blocked. In addition, p21WAF1/CIP1/SDI1 also blocks DNAsion of cyclin E. Four cell lines (JHH-4; HLE; HuH-7;replication depending on proliferating cell nuclear anti-PLC/PRF/5) deficient of p21WAF1/CIP1/SDI1 protein did not

overexpress cyclin E protein. As to expression of the gen (PCNA), resulting in G1 arrest.other cell-cycle-related proteins, cyclin A, cyclin D1, Recent studies have shown that abnormalities inCDK2 or CDK4, no significant difference was detected cyclins generally lead to loss of growth control (1–3).among the 12 cell lines. These findings indicate that Gene amplification and/or overexpression of cyclin D1the human liver tumor cell lines which have the have been found in various human tumors, such asp21WAF1/CIP1/SDI1-inducible barriers of the cell cycle pro- parathyroid adenoma, mantle-cell lymphoma, breastgression can go through the G1/S checkpoint by over- cancer, squamous-cell carcinoma, and hepatocellularexpressing cyclin E. q 1998 Academic Press carcinoma (4,5). Similarly, gene amplification and over-

expression of cyclin E have been observed in colorectalcarcinoma (6), gastric carcinoma (7), breast cancer (8),

Progression of the cell cycle in eukaryotic cells is ovarian cancer (9) and acute lymphoblastic leukemiaregulated by a series of serine/threonine protein ki- (10). This cyclin may also work as a potential prognos-nases which consist of a catalytic subunit, cyclin depen- tic marker for breast cancer because the levels of itsdent kinase (CDK), and a regulatory subunit, cyclin expression frequently increase with the tumor grade(for reviews, see Refs. 1–3). G1 cyclins regulate the and stage (11).entry of cells into S phase through G1. D-type cyclins About 70% of hepatocellular carcinomas (HCCs) do(D1, D2 and D3) act at mid-G1 in the cell cycle with not show any p53 alterations (12). It means that p53CDK4 or CDK6. Cyclin E acts at late-G1 by forming a which functions normally can induce expression ofcomplex with CDK2. The ectopic expression of cyclin p21WAF1/CIP1/SDI1. This raises a question as to how tumorD1 or cyclin E causes a shortening of the G1 phase. cells proliferate continuously regardless of expressionConversely, inhibition of cyclin D1 activity with its an- of p21WAF1/CIP1/SDI1 CDK inhibitor. Here we describe atibody or antisense RNA leads to G1 arrest. Two pro- relationship between p21WAF1/CIP1/SDI1 and cell-cycle-re-teins of the retinoblastoma (Rb) family, Rb and p107, lated proteins in 12 human liver tumor cell lines, and

discuss the role of cyclin E overexpression in thecell cycle progression of the cell lines expressing1 Corresponding author. Fax: (/81) 86-235-7400. E-mail:

mnamba@med.okayama-u.ac.jp. p21WAF1/CIP1/SDI1 protein.

0006-291X/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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FIG. 1. Expression of cell-cycle-related proteins and pRb kinase activity associated with CDK2 and CDK4 in human liver tumor celllines. Protein samples from proliferating cells (40 mg protein per lane) were analyzed by Western blot with the indicated polyclonal antibodies,such as anti-p21WAF1/CIP1/SDI1 (A), anti-CDK2 (D), and anti-cyclin E (E). In addition, protein extracts (100 mg protein per lane) were treatedwith anti-CDK2 (B) or anti-CDK4 (C) antibodies, and the immunoprecipitates were analyzed for kinase activity with a pRb fragment as asubstrate. Normal human fibroblasts (OUMS-24) which were serum-starved for 1 week were used as a negative control. W: wild-type p53;M: mutant-type p53.

cyclin E (Santa Cruz Biotechnology, CA) or cyclin A (affinity-chro-MATERIALS AND METHODSmatographically purified rabbit IgG, produced in our laboratory).After washing, the membranes were incubated for 1 hr with a 1:1000Cell culture. HCC cell lines, JHH-1, JHH-2, JHH-4, JHH-5, JHH-diluted second antibody, horseradish peroxidase-conjugated sheep6, JHH-7, Hep3B, HLE, HuH-7, PLC/PRF/5, and hepatoblastomaanti-rabbit IgG (MBL, Nagoya, Japan), and the reaction products(HB) cell lines, HuH-6 Clone 5 and HepG2, were cultured in Dulbec-were visualized using an enhanced chemiluminescence system (ECL,co’s modified Eagle’s medium (DMEM, Nissui, Tokyo, Japan) supple-Amersham). The signal intensity was quantified using Image Quantmented with 10% fetal bovine serum (FBS) and 100 mg/ml kanamy-version 3.3 (Molecular Dynamics, CA).cin. Characteristics of these cell lines have already been described

elsewhere (13, 14). Rb kinase assay. Cells were treated with a hypotonic buffer [50mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM EGTA, 1 mM sodiumSouthern blot analysis. Genomic DNA was extracted from cellsfluoride, 0.1 mM sodium orthovanadate, 200 mg/ml PMSF, 2 mg/mlby the phenol method, digested with the restriction endonucleaseaprotinin and 2 mg/ml leupeptin], and centrifuged. The pellets werePst I, electrophoresed on a 1% agarose gel, and transferred by thethen resuspended in a nuclear extraction buffer [50 mM Tris-HClcapillary method to a Hybond N/ membrane (Amersham, London,(pH 8.0), 150 mM NaCl, 0.1% Tween-20, 1 mM EDTA, 1 mM EGTA,UK). The membrane was hybridized at 427C with a probe in a solu-1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 200 mg/mltion [51 SSC, 50% formamide, 11 Denhart’s solution, 20 mM sodiumPMSF, 2 mg/ml aprotinin and 2 mg/ml leupeptin]. Immunoprecipita-phosphate (pH 6.8), 5 mM EDTA, 0.2% SDS and 100 mg/ml shearedtion was carried out with 1 mg of the indicated antibody (againstheat-denatured salmon sperm DNA]. Human cyclin E cDNA probeCDK2 or CDK4), and the immunocomplexes were recovered withwas labeled with [a-32P] dCTP by a random priming reaction.protein A-Sepharose. For the Rb kinase assay, the immunocomplexes

Northern blot analysis. Total cellular RNA was extracted from bound to the protein A-beads were washed three times with thecell cultures in a log-phase by the guanidinium thiocyanate-phenol nuclear extraction buffer, and twice with a kinase buffer [50 mMmethod. Total cellular RNA (20 mg) was fractionated by electrophore- Tris-HCl (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA andsis through a formaldehyde-agarose gel, and transferred to the Hy- 1 mM dithiothreitol]. Finally, the pellets were resuspended in 30 ml ofbond N/ membrane. Hybridization was performed as described above the kinase buffer supplemented with a substrate (0.4U Rb fragment,for Southern blot. Pharmacia, Uppsala, Sweden) and 10 mCi of [g-32P] ATP, and were

incubated for 30 min at 307C. The reaction was stopped by addingWestern blot analysis. Logarithmically growing cells were15 ml of 3 1 Laemmli sample buffer. The reaction mixtures werewashed twice with ice-cold PBS and lysed in a lysis buffer [50 mMthen run on a 12% SDS polyacrylamide gel, and the phosphorylatedTris-HCl (pH 8.0), 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTAsubstrates were visualized by autoradiography.(pH 8.0), 1 mM EGTA (pH 8.0), 1 mM sodium fluoride, 0.1 mM

sodium orthovanadate, 1 mM dithiothreitol, 200 mg/ml phenylmeth-ylsulfonylfluoride (PMSF), 2 mg/ml aprotinin and 2 mg/ml leupeptin]. RESULTSCell lysates were centrifuged at 12,000 1 g for 30 min at the 47Cand supernatants were separated. Protein concentration was deter-

Status of the p53 gene and expression ofmined by the method of Bradford (Bio-Rad, Hercules, CA). Totalp21WAF1/CIP1/SDI1. The status of the p53 gene of 12 hu-protein (40 mg) was diluted with 3 1 Laemmli sample buffer, incu-

bated at 657C for 15 min, fractionated by SDS polyacrylamide gel man liver tumor cell lines is shown in Figure 1. Thiselectrophoresis, and blotted onto a nitrocellulose membrane (Amer- was previously determined by functional analysis ofsham). The membranes were incubated for 1 hr with TBST [20 mM separated p53 alleles in yeast (FASAY) assay (15).Tris-HCl (pH 7.2), 100 mM NaCl, 0.1% Tween 20] containing 5%

Based on these findings, we examined a relationshipdried milk, and then for 1.5 hr with a 1:1000 dilution of primaryantibodies against p21WAF1/CIP1/SDI1, Rb, CDK2, CDK4, cyclin D1, between the p53 status and p21WAF1/CIP1/SDI1 expres-

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FIG. 2. Status of the cyclin E gene and expression levels of cyclin E mRNA in human liver tumor cell lines. (A) 10 mg genomic DNAdigested with PstI was hybridized with 32P-labeled cDNA for cyclin E, as described in MATERIALS AND METHODS. (B) 20 mg total RNAwas hybridized with 32P-labeled cyclin E cDNA probe (top), or with 32P-labeled GAPDH cDNA probe (middle) which was used for an internalcontrol. The autoradiographic signal density was quantified by Image-Quant system. Ratio of cyclin E mRNA per GAPDH mRNA of 12 celllines is shown by arbitrarily setting an unit of normal human fibroblasts OUMS-24 as 1.0 (bottom).

sion in the 12 human liver tumor cell lines. The cell lines. Consequently, a significant difference wasdetected in the expression of cyclin E between thep21WAF1/CIP1/SDI1 protein was detected in all the cell lines

expressing the wild-type p53 (JHH-1, HuH-6, HepG2, p21WAF1/CIP1/SDI1-expressing and -deficient cell lines. Asshown in Figure 1E, the cyclin E protein was overex-Hep3B, and JHH-7), but not in four (HLE, HuH-7,

PLC/PRF/5, and JHH-4) out of seven cell lines express- pressed in seven cell lines (JHH-1, HepG2, Hep3B,JHH-7, JHH-2, JHH-6, and JHH-5) out of eight celling mutant-type p53. However, regardless of expres-

sion of the mutant-type p53, the three cell lines (JHH- lines expressing p21WAF1/CIP1/SDI1 protein, althoughone of them (JHH-5) overexpressed a reduced size of2, JHH-5, and JHH-6) expressed the p21WAF1/CIP1/SDI1

(Fig. 1A). Thus, there was no relationship between the cyclin E. The rest (HuH-6) of the 8 cell lines withp21WAF1/CIP1/SDI1 showed a decreased expression of cyclinp53 gene status and the expression of p21WAF1/CIP1/SDI1.

Then we measured kinase activities of CDK2 and E. On the other hand, it was weakly expressed in the 4cell lines deficient of the p21WAF1/CIP1/SDI1 protein (HLE,CDK4 by the phosphorylation assay using an Rb frag-

ment as a substrate. There was no significant differ- HuH-7, PLC/PRF/5, and JHH-4).The levels of cyclin D1 protein in the 12 human liverence in the activities of CDK2 and CDK4 among the

12 liver tumor cell lines (Fig. 1B, C). In other words, tumor cell lines were almost the same as that in thenormal human fibroblast cell strain OUMS-24 (dataCDK2 and CDK4 in all the cell lines were active inde-

pendently of the presence or absence of p21WAF1/CIP1/SDI1 not shown), although this cyclin has been reported tobe overexpressed in several carcinomas, including hep-CDK inhibitor. In addition, Western blot analysis with

CDK2 antibody showed a phosphorylated form of atoma (4, 5). In addition, there was no significant differ-ence in the expression of cyclin A, CDK2, or CDK4CDK2 (active form, faster band) in all the liver tumor

cell lines (Fig. 1D). proteins between the p21WAF1/CIP1/SDI1-expressing and-deficient cell lines (data not shown). PhosphorylationOverexpression of cyclin E in liver tumor cell linesof Rb protein was detected at similar levels betweenwith p21WAF1/CIP1/SDI1. The above findings led us to athese cell lines, although the Rb protein was lost inquestion as to how liver tumor cells activate CDKs inHLE and Hep3B cells (data not shown).the presence of p21WAF1/CIP1/SDI1 CDK inhibitor. Thus we

examined the expression of several kinds of the cell- Mechanisms of overexpression of cyclin E protein.We examined the mechanisms by which cyclin Ecycle-related proteins, such as cyclin A, cyclin D1,

cyclin E, CDK4, and Rb, in these human liver tumor was overexpressed in the cell lines having the

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p21WAF1/CIP1/SDI1 protein. We considered three possibili-ties: gene amplification, transcriptional abnormalityand proteolytic abnormality. First, Southern blot anal-ysis on the Pst I-digested genomic DNA from the 12human liver tumor cell lines and from normal humanfibroblasts (OUMS-24) showed no difference in the sizeor intensity of cyclin E bands (Fig. 2A). Thus, it is obvi-ous that overexpression of cyclin E in the cell lineswith the p21WAF1/CIP1/SDI1 protein is not due to the geneamplification reported in various human cancers, suchas breast cancer, ovarian cancer, colorectal carcinomaand gastric carcinoma (6–9). Second, Northern blotanalysis showed increased mRNA expression of cyclinE in most of the cell lines having p21WAF1/CIP1/SDI1, suchas JHH-1, HepG2, Hep3B, JHH-7 and JHH-2 (Fig. 2B).Third, if mutation and/or deletion may have occurredin the PEST sequence in the cyclin E gene, the half-life of the cyclin E protein increases (1, 16). However,all the liver tumor cell lines showed the wild-type PESTsequence (data not shown). Taken together, these find-ings demonstrate that overexpression of cyclin E in theliver tumor cell lines with p21WAF1/CIP1/SDI1 protein isdue to the enhanced transcription of the gene.

Abnormal expression of cyclin E throughout the cellcycle. In general, cyclin E is maximally expressed atthe G1-S transition in normal cells (17, 18). To deter- FIG. 3. Time course kinetics of cyclin E expression in JHH-1 cells

with p21WAF1/CIP1/SDI1, and HLE cells deficient of p21WAF1/CIP1/SDI1. Totalmine whether the levels of cyclin E protein would varyprotein was extracted from serum-starved and serum-restimulated cul-during the cell cycle, we synchronized the typical twotures of JHH-1 or HLE cells. Western blot analysis was carried outcell lines, i.e., the p21WAF1/CIP1/SDI1-expressing JHH-1 using a polyclonal antibody against cyclin E. The intensity of each band

and -deficient HLE cell lines by serum starvation. The was quantified by the Image-Quant system (s: JHH-1, l: HLE).expression levels of cyclin E protein did not change inJHH-1 cells during the 48-hour culture, while in HLEcells, it peaked at 12 hours and then gradually de-

is a discrepancy between the previously results andcreased with time (Fig. 3). Since the population dou-ours. This suggests that the cyclin E gene expressionbling time of JHH-1 cells is 44-50 hours (13), it is obvi-is induced not only via the E2F1-pathway but also viaous that overexpression of cyclin E in the cells is inde-other pathways such as the myc-pathway (21). Fur-pendent of the cell cycle. Taken together, JHH-1 cellsthermore, cyclin E was overexpressed in JHH-1 cellsmay have abnormalities not only in the expression lev-throughout the cell cycle. This unscheduled manner ofels but also in the expression time of cyclin E in thecyclin E expression has also been observed in severalcell cycle.other tumor cell lines such as promyelocytic leukemiaHL-60, histiocytic lymphoma U937, ductal breast carci-

DISCUSSION nomas T47D and Hs 587T, and colon carcinoma Colo320DM (22). Thus, cyclin E may interact with CDK2throughout the cell cycle, thereby phosphorylating aThis study shows that the human liver tumor cell

lines expressing p21WAF1/CIP1/SDI1 CDK inhibitor overex- variety of substrates, bypassing some growth regula-tory mechanisms, and accelerating the cell cycle. Thesepressed cyclin E. Recent reports indicate that tran-

scription of the cyclin E gene is up-regulated by E2Fs abnormalities may play an important role in hepatocar-cinogenesis.(19, 20) and down-regulated by pRb (20). The cyclin E

gene expression is tightly regulated in rat fibroblasts There was no relationship between the p53 gene sta-tus and the p21WAF1/CIP1/SDI1 expression in 12 humanwith an accumulation in G1 of the cell cycle, similar to

the kinetics of E2F1 (19). In the present study, E2F1 liver tumor cell lines tested. p21WAF1/CIP1/SDI1 is knownto be induced via the p53 dependent (23) and/or inde-protein level did not change in 12 liver tumor cell lines

and was not in proportion to the cyclin E expression pendent pathways (24, 25). In case of the p53-depen-dent manner, p21WAF1/CIP1/SDI1 expression is induced by(data not shown). In addition, cyclin E mRNA was over-

expressed in JHH-1 cells, regardless of the presence of radiation- or chemically-induced DNA damage (26, 27).In addition, p21WAF1/CIP1/SDI1 expression is also inducedpRb, a negative regulator (data not shown). Thus, there

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10. Scuderi, R., Palucka, K. A., Pokrovskaja, K., Bjorkholm, M., Wi-by up-regulation of p53 activity by overexpression ofman, K. G., and Pisa, P. (1996) Blood 87, 3360–3367.cyclin E (28, 29). Expression of p21WAF1/CIP1/SDI1 protein

11. Keyomarsi, K., O’Leary, N., Molnar, G., Lees, E., Fingert, H. J.,in the cell lines with the wild-type p53 (JHH-1, HuH-and Pardee, A. B. (1994) Cancer Res. 54, 380–385.6, HepG2, Hep3B, and JHH-7) may be due to the up-

12. Oda, T., Tsuda, H., Scarpa, A., Sakamoto, M., and Hirohashi, S.regulation of p53 activity by overexpression of cyclin (1992) Cancer Res. 52, 6358–6364.E. On the other hand, p21WAF1/CIP1/SDI1 protein may be 13. Homma, S. (1985) Jikeikai Med. J. 32, 289–315.induced in JHH-2, JHH-5, and JHH-6 cells via the p53- 14. Miyazaki, M., and Namba, M. (1994) in Atras of Human Tumorindependent pathway, since these cell lines have the Cell Lines (Hey, R. J., Park, J-G., and Gazdar, A., Eds.), pp. 185–

212, Academic Press, San Diego, CA.mutant-type p53. Although the mechanisms by which15. Mihara, K., Miyazaki, M., Kondo, T., Fushimi, K., Tsuji, T., In-p21WAF1/CIP1/SDI1 is induced in these cell lines are un-

oue, Y., Fukaya, K., Ishioka, C., and Namba, M. (1997) Actaclear, it has been recently reported that p21WAF1/CIP1/SDI1

Med. Okayama 51, 261–265.can be induced by some transcriptional factors, such as16. Won, K-A., and Reed, S. I. (1996) EMBO J. 15, 4182–4193.Myo D (30), IRF1 (31), STAT1 (32), and E2F (33). Taken17. Duli’c, V., Lees, E., and Reed, S. I. (1992) Science 257, 1958–together, p21WAF1/CIP1/SDI1 induction in some liver tumor

1961.cell lines with the mutant-type p53 may also be due to

18. Ohtsubo, M., and Roberts, J. M. (1993) Science 259, 1908–1912.the these transcriptional factors.

19. Ohtani, K., DeGregori, J., and Nevins, J. R. (1995) Proc. Natl.Lin et al (34) reported that transfection of Acad. Sci. USA 92, 12146–12150.

p21WAF1/CIP1/SDI1 cDNA greatly suppressed the growth 20. Geng, Y., Eaton, E. N., Picon, M., Roberts, J. M., Lundberg, A. S.,of p53-defective cell lines, Saos-2 and H1299. However, Gifford, A., Sardet, C., and Weinberg, R. A. (1996) Oncogene 12,

1173–1180.cotransfection of cyclin D1 or cyclin E cDNA with21. Perez-Roger, I., Solomon, D. L., Sewing, A., and Land, H. (1997)p21WAF1/CIP1/SDI1 cDNA partially recovered the prolifera-

Oncogene 14, 2373–2381.tion activity of the cells from the growth arrest caused22. Gong, J., Ardelt, B., Traganos, F., and Darzynkiewicz, Z. (1994)by p21WAF1/CIP1/SDI1. These findings suggest that there

Cancer Res. 54, 4285–4288.is a certain threshold of p21WAF1/CIP1/SDI1 above which23. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Per-cells can not enter the S phase of the cell cycle. In other sons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and

words, p21WAF1/CIP1/SDI1 causes G1 arrest only when the Vogelstein, B. (1993) Cell 75, 817–825.amount of this CDK inhibitor stoichiometrically ex- 24. Michieli, P., Chedid, M., Lin, D., Pierce, J. H., Mercer, W. E.,ceeds that of the cyclin-CDK complexes. These findings and Givol, D. (1994) Cancer Res. 54, 3391–3395.indicate that up-regulation of cyclin E expression 25. Zang, H., Hannon, G. J., and Beach, D. (1994) Genes Dev. 8,

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Harper, J. W., Elledge, S. J., and Reed, S. I. (1994) Cell 76, 1013–1023.

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