Inhibitory effect of mimosine on proliferation of human lung cancer cells is mediated by multiple mechanisms

Cancer Letters 145 (1999) 1±8 www.elsevier.com/locate/canlet

Inhibitory effect of mimosine on proliferation of human lung cancer cells is mediated by multiple mechanisms Hui-Chiu Chang a, Te-Hsiu Lee b, Lea-Yea Chuang c, Ming-Hong Yen d, Wen-Chun Hung e,* a

Department of Physiology, Kaohsiung Medical College, Kaohsiung 807, Taiwan Graduate Institute of Medicine, Kaohsiung Medical College, Kaohsiung 807, Taiwan c Department of Biochemistry, Kaohsiung Medical College, Kaohsiung 807, Taiwan d School of Pharmacy, Kaohsiung Medical College, Kaohsiung 807, Taiwan e School of Technology for Medical Sciences, Kaohsiung Medical College, Kaohsiung 807, Taiwan b

Received 10 March 1999; received in revised form 28 April 1999; accepted 13 May 1999

Abstract The plant amino acid mimosine has been reported to block cell cycle progression in the late G1 phase. A recent study showed that mimosine might induce growth arrest by activating the expression of p21 CIP1, a cyclin-dependent kinase inhibitor (CDKI), and by inhibiting the activity of cyclin E-associated kinases in human breast cancer cells. However, mimosine at higher concentrations also blocked proliferation of p21 2/2 cells by unknown mechanisms. In this study, we investigated the effect of mimosine on the expression of cyclins and CDKIs in human lung cancer cells. We found that mimosine speci®cally inhibited cyclin D1expression in H226 cells. The expression of another G1 cyclin, cyclin E, was not regulated by mimosine in all lung cancer cell lines examined. Moreover, mimosine induced p21 CIP1 expression in H226 and H358 cells, while it activated p27 KIP1 expression in H322 cells. However, mimosine does not affect transcription of these genes directly because signi®cant changes in cyclin D1 or CDKI expression were observed at 12±24 h after drug addition. Our results indicate that mimosine may block cell proliferation by multiple mechanisms and this amino acid is a useful agent for the study of cell cycle control. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mimosine; Cyclin D1; Cyclin-dependent kinases; Cyclin-dependent kinase inhibitors; Lung cancer

1. Introduction l-Mimosine, a non-protein plant amino acid derived from seeds of Leucaena leucocephala or Mimosa pudica, was reported to be a speci®c and reversible late-G1 blocker of the cell cycle [1,2].

* Corresponding author. Fax: 1886-7-751-2369.

This drug is used as an effective synchronizing agent for investigating DNA replication and cell cycle progression in mammalian cells [3,4]. However, the molecular mechanism of the action of mimosine is not fully de®ned at present. Mimosine was reported to inhibit DNA synthesis by altering iron and deoxyribonucleotide triphosphate metabolism [5,6]. On the other hand, this drug may block cell cycle progression by interfering with the function of proteins which are

0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00209-8

2

H.C. Chang et al. / Cancer Letters 145 (1999) 1±8

involved in the processes of DNA replication. The proteins which are affected by mimosine include serine methyltransferase, ribonucleotide reductase and eukaryotic initiation factor 5A [7±9]. A recent study have demonstrated that the progression of the cell cycle is governed by cyclins and cyclin-dependent kinases (CDKs) [10]. Mammalian G1 cyclins include three D-type cyclins (D1±3) and cyclin E [11,12]. These cyclins associated with their catalytic subunits CDK4, 6 and 2 to phosphorylate retinoblastoma proteins (pRb) and to promote the progression of cell cycle from G1 to S phase [13,14]. On the contrary, the progression of cell cycle through S, G2 and M phase is controlled by the A- and B-type cyclins and CDK2 [15,16]. The study of cell cycle control becomes more complicated because of the identi®cation of a group of negative regulatory proteins that bind to cyclin-CDK complexes and inhibit their catalytic activity [17]. These proteins, termed CDK inhibitors (CDKIs), are classi®ed into two groups based on their protein sequence similarities and putative CDK targets. The ®rst class is the INK4 family (including p15 INK4B, p16 INK4A, p18 INK4C and 19 INK4D) which speci®cally inhibit the activity of the cyclin D-associated kinases CDK4 and CDK6 [18,19]. The second class is the CIP/KIP proteins (including p21 CIP1, p27 KIP1 and p57 KIP2) which have broad speci®city and inhibit the activity of most cyclin-CDK complexes [20,21]. It is now known that the positive effectors (cyclins and CDKs) and negative effectors (CDKIs) described above may interact with each other to control the progression of cell cycle [22,23]. Because mimosine arrests cell cycle progression at a speci®c point in the cycle, it is of interest to study whether this drug modulates the expressions of cyclins or CDKIs. A very recent report demonstrated that mimosine upregulates the expression of p21 CIP1 through a p53-independent pathway [24]. However, because cell growth of p21 2/2 cells could still be inhibited by mimosine at high concentration, it is possible that other cell cycle regulatory proteins are involved in mimosine-triggered G1 arrest. Therefore, we examine the effect of mimosine on the expression of G1 cyclins and CDKIs and the activity of CDKs in p53-defective lung cancer cell lines. Our results indicate that G1 growth arrest induced by mimosine is mediated by different mechanisms.

2. Materials and methods 2.1. Materials Antibodies against cyclin D1 and p21 CIP1 were obtained from Calbiochem (San Diego, CA) and anti-human cyclin E, p27 KIP1, p57 KIP2 and GST-RB fusion protein were obtained from Santa Cruz, CA. Mimosine, 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and other chemicals were obtained from Sigma (St. Louis, MO). [g± 32P]ATP was purchased from Amersham (Buckinghamshire, UK). 2.2. Cell culture and treatment Non-small cell lung cancer cell lines H226, H322 and H358 were cultured in DMEM/F12 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) in a 5% CO2 incubator at 378C. Cells were grown in 10% FCS medium to subcon¯uence and then incubated in 10% FCS medium containing different concentrations of mimosine for different times and were harvested for analysis. 2.3. Cell cycle analysis by ¯ow cytometry Cells were trypsinized, washed with phosphatebuffered saline (PBS) and ®xed in 95% ethanol at 2208C for 1 h. Following centrifugation, cells were resuspended in 400 ml of PBS containing 1 mg/ml RNase A and 0.5% Triton X-100 and incubated at room temperature for 1 h. Cells were then stained with 50 mg/ml of propidium iodide for 30 min. Fluorescence emitted from the propidium iodide-DNA complexes was analyzed by FACScan ¯ow cytometry and cell cycle distribution was calculated with the CellFit software as described previously [25]. 2.4. Immunoblotting After treatment, cells were washed with ice-cold PBS and harvested in a lysis buffer (50 mM Tris± HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl ¯uoride (PMSF), 2 mg/ml pepstatin A, 2 mg/ml leupeptin and 1 mg/ml aprotinin) for 20 min on ice and centrifuged at 12 000 £ g for 20 min. Protein concentrations of the cell lysates were

H.C. Chang et al. / Cancer Letters 145 (1999) 1±8

3

Table 1 Effect of mimosine on cell proliferation of lung cancer cells analyzed by MTT assay a

blots were developed by using the ECL chemiluminescence system (Amersham).

Treatment

2.5. Immune complex kinase assay

No additive 100 mM mimosine 400 mM mimosine

Viable cells (% of control) H226

H322

H358

100 50.8 ^ 4.2 39.7 ^ 3.8

100 72.8 ^ 5.6 58.8 ^ 7.3

100 95.2 ^ 3.4 60.2 ^ 6.0

a Cells were incubated without or with different concentrations of mimosine at 378C for 48 h and the effect of mimosine on cell growth was examined by MTT assays. The percentage of cell proliferation was calculated by de®ning the absorption of cells without treatment of mimosine as 100% and all determinations were done in three replicates.

determined by using a BCA protein assay kit (Rockford, IL). The cell lysates (50 mg protein/sample) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were then transferred to nitrocellulose membranes. The blots were blocked in 5% non-fat milk in TBST (20 mM Tris±HCl (pH 7.4), 137 mM NaCl and 05% Tween-20) overnight at 48C and probed with various antibodies for 2 h at room temperature. After extensively washing in TBST buffer, peroxidase-conjugated secondary antibody was added in blocking buffer for another 1 h and the

Table 2 Effect of mimosine on cell cycle progression of lung cancer cells assayed by ¯ow cytometric analysis a Cell line

H226 Control Mimosine H322 Control Mimosine H358 Control Mimosine a

Cell cycle distribution (%) G1

S

G2/M

51.8 86.2

28.7 6.8

19.5 7.0

60.3 79.8

25.2 10.4

14.5 9.8

64.7 77.1

23.7 12.3

11.6 10.6

Cells were cultured in the absence or presence of 400 mM mmosine for 48 h. After incubation, cells were harvested, stained and subjected to ¯ow cytometric analysis. The data from a typical experiment are shown and another two independent experiments gave similar results.

Cell extracts were prepared as described above and 500 mg of proteins were subjected to immunoprecipitation with anti-cyclin D1 antibody. The immunocomplexes were collected with protein G-agarose at 48C for 1 h and were washed four times with lysis buffer and twice with kinase buffer (50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 and 1 mM dithiothreitol). Immunoprecipitates were resuspended with 30 ml of kinase buffer containing 1 mg GST-Rb, 60 mM cold ATP and 10 mCi [g± 32P]ATP and incubated at room temperature for 30 min. The reactions were stopped by the addition of Laemmli sample buffer and boiling for 10 min. The phosphorylated proteins were electrophoresed on 10% SDS-PAGE gels. The gels were stained, destained, dried and then exposed to Kodak X-ray ®lms. The phosphorylated GST-Rb bands were excised from the gels and the amount of radioactivity incorporated into the GST-Rb proteins was determined by scintillation counting. Results obtained from three independent experiments were expressed as mean ^ standard error. 2.6. Growth inhibition assays Cells (2 £ 103 cells/well) were grown in 96-well plates in 10% FCS medium and then cultured in different concentrations of mimosine for 48 h. After incubation, 50 ml of MTT reagents (100 mg) were added to each well and allowed to incubate for 4 h at 378C. Cells were then solubilized in 200 ml dimethyl sulfoxide with absorbance measured at 540 nm by using a microplate reader (Molecular Probe). 3. Results 3.1. Mimosine induces G1 growth arrest in human lung cancer cells To evaluate the inhibitory effect of mimosine on the proliferation of human lung cancer cells, exponentially growing cancer cells cultured in complete medium were incubated in different concentrations of mimosine for 48 h and cell growth was determined

4

H.C. Chang et al. / Cancer Letters 145 (1999) 1±8

Conversely, the protein levels of cyclin E in these cell lines were not noticeably altered by mimosine. 3.3. Mimosine differentially regulates the expression of cyclin-dependent kinase inhibitors in different lung cancer cells Fig. 1. Modulation of the expression of G1 cyclins by mimosine in human lung cancer cells. Cells were incubated without or with 400 mM mimosine for 48 h and then harvested in a lysis buffer. Equal amounts of cellular proteins were subjected to SDS-polyacrylamide gel electrophoresis and the expression of G1 cyclins (D1 and E) was examined by Western blot analysis.

by a MTT-based assay. As shown in Table 1, mimosine inhibited the proliferation of H226, H322 and H358 cells in a dose-dependent manner and this drug, at 400 mM blocked cell growth by 40±60% in the lung cancer cell lines studied. We further investigated the effect of mimosine on cell cycle progression of these cells by ¯ow cytometric analysis. In accordance with the results of other studies, mimosine induced G1 growth arrest in lung cancer cells (Table 2). The percentages of cells in G1 phase of the cell cycle in H226, H322 and H358 cells cultured in complete medium were 52, 60 and 65%, respectively. After incubation of 400 mM mimosine for 48 h, the percentages of cells in G1 phase increase to 77±86 % in these cell lines. 3.2. Mimosine differentially modulates the expression of G1 cyclins in different lung cancer cell lines Since mimosine effectively blocks cell cycle progression at the G1 phase, we investigated whether this drug can modulate the expression of G1 cyclins (D1 and E) in these cells. Cell were cultured in the absence or presence of 400 mM mimosine in 10% FCS medium for 48 h, whole cell lysates were prepared, subjected to SDS-PAGE and analyzed by immunoblotting. As shown in Fig. 1, Western blot analysis with anti-cyclin D1 and anti-cyclin E antibodies con®rms the presence of these cyclins in the cell extracts and all these three lung cancer cell lines expressed high levels of cyclin D1 and E proteins. We found that mimosine inhibited that expression of cyclin D1 in H226 cells, but this drug did not block cyclin D1 expression in another two cell lines.

Because mimosine induces G1 growth arrest without inhibition of the expression of G1 cyclins in H322 and H358 cells, it is possible that this drug acts via other molecular mechanisms to block cell growth in these cells. A previous study reported that mimosine activated the expression of p21 CIP1, a cyclin-dependent kinase inhibitor (CDKI) in 21PT breast cancer cells [24]. Therefore, we investigated whether this drug modulated the expression of the CIP/KIP gene family in lung cancer cells. As shown in Fig. 2, mimosine induced p21 CIP1 expression in H226 and H358 cells but not in H322 cells. It should be noted that the p53 gene in these cell lines was inactivated by point mutation or homozygous deletion [26]. Therefore, the activation of p21 CIP1 gene expression by mimosine in H226 and H358 cells is independent of p53. On the contrary, the induction of p27 KIP1 by this drug was only detected in H322 but not in the other two cell lines and the expression of p57 KIP2 was not signi®cantly regulated by mimosine in all three cell lines. This is a new ®nding, that mimosine may activate p27 KIP1 in some cancer cell lines. 3.4. Down-regulation of cyclin D1 or induction of CDKIs by mimosine leads to inhibition of cyclin D1associated kinase activity Previous studies have demonstrated that cyclin

Fig. 2. Induction of the CIP/KIP gene family by mimosine in lung cancer cells. Cells were incubated in the absence or presence of 400 mM mimosine for 48 h and the expression of p21 CIP1, p27 KIP1 and p57 KIP2 was investigated by Western blot analysis.

H.C. Chang et al. / Cancer Letters 145 (1999) 1±8

5

were inhibited by 56, 38 and 40%, respectively after mimosine incubation (Fig. 3). Additionally, we also found that the magnitude of inhibition of proliferation by mimosine was associated with the magnitude of inhibition of cyclin D1-associated kinase activity by this drug. Inhibition of cyclin D1 and induction of p21 CIP1 were simultaneously induced by mimosine in H226 cells, therefore cyclin D1-associated kinase activity and cell growth in H226 cells were blocked to a greater extent than that of another two cell lines (Table1, Fig.3). Thus, down-regulation of cyclin D1 expression or up-regulation of the expression of the CIP/KIP gene family provides different mechanisms that mediate the growth-inhibitory effect of mimosine in lung cancer cells.

Fig. 3. Inhibition of cyclin D1-associated kinase activity by mimosine in lung cancer cells. Cyclin D1-associated kinase activity was examined by in vitro kinase assays. The cyclin D1/CDKcomplexes were collected by sequential addition of anti-cyclin D1 and proteinG agarose and the activity of cyclin D1-associated kinases was determined by the incorporation of 32P into the kinase substrate, GST-Rb fusion protein. The phosphorylated GST-Rb bands were excised from the gels and the amounts of radioactivity incorporated into the GST-Rb proteins was determined by scintillation counting. Results from three independent experiments were expressed as mean ^ SEM. Untreated cells (B); Mimosine-treated cells (g).

D/CDK4 or cyclin D/CDK6 complexes may phosphorylate the retinobloastoma protein (pRb) to inactivate their inhibitory effect of cell cycle progression [27,28]. We examined whether the inhibition of cyclin D1 or induction of CDKIs by mimosine may lead to attenuated cyclin D1-associated kinase activity. The cyclin D1-associated kinase activity was analyzed by a immuno-precipitation/in vitro kinase assay technique. After drug treatment, cyclin D1/CDKs complexes were collected by the sequential addition of anti-cyclin D1 antibody and protein G-agarose. Our results showed that the incorporation of 32P into GSTRb (a fusion protein substrate for cyclin D1-associated kinases) was reduced in H226, H322 and H358 cells after treatment of 400 mM mimosine for 48 h. The amount of radioactivity incorporated into the GST-Rb proteins was determined by scintillation counting and the results from three independent experiments were averaged. We found that the cyclin D1-associated kinase activities of H226, H322 and H358 cells

3.5. Kinetic study of alteration of cyclin D1 and CDKI expression induced by mimosine An important issue to be clari®ed is whether mimosine directly affects the transcription of cyclin D1 or CDKI genes in lung cancer cells. To answer this question we studied the kinetics of mimosine-induced alteration of gene expression in H226 and H322

Fig. 4. Kinetic study of alteration of cyclin D1 and CDKI expression Induced by mimosine. H226 and H322 lung cancer cells were treated with 400 mM mimosine for different time intervals and harvested by a lysis buffer. The protein levels of cyclin D1, p21 CIP1 and p27 KIP1 were detected by Western blot analysis. (A) change of cyclin D1 and p21 CIP1 expression in mimosine-treated H226 cells. (B) mimosine-induced p27 KIP1 expression in H322 cells is time-dependent.

6

H.C. Chang et al. / Cancer Letters 145 (1999) 1±8

cells. Because mimosine had been demonstrated to arrest DNA replication within 2 h of addition, we examined the protein levels of cyclin D1 or CDKI at 1, 12 and 24 hours after drug addition. As shown in Fig. 4A, down-regulation of cyclin D1 expression was found at 24 h after mimosine treatment in H226 cells. Similarly, signi®cant induction of p21 CIP1 in H226 cells or p27 KIP1 in H322 cells was observed at 12±24 h after drug addition (Fig. 4A,B). Therefore, it seems that mimosine does not affect the transcription of these genes directly. 4. Discussion Modulation of the expression of cyclins and the kinase activity of CDKs by mimosine is studied in human lung cancer cells. We found that mimosineinduced growth inhibition was mediated by multiple mechanisms. Firstly, mimosine decreased cyclin D1 protein levels and inhibits cyclin D1-associated kinase activity in H226 cells. Cyclin D1 is encoded by the CCND1/PRAD1 gene on chromosome 11q13 and is reported to be deregulated in many types of human malignancies [29±31]. However, regulation of the expression of cyclin D1 was not fully understood until now. Several regulatory elements are identi®ed in the promoter region of this cyclin, these include the E-box (Myc/Max binding site), E2F, Sp1 and CREB/ ATF binding sites [32]. Additionally, several upstream signaling pathways (such as the p42/p44 MAPK pathway) have been implicated in the modulation of cyclin D1 expression [33]. The signaling pathway which mimosine used to block the expression of cyclin D1 is not known at present. A recent study showed that this drug might inhibit cyclin D1 expression by depriving intracellular iron levels in MDA-MB-453 breast cancer cells [34]. However, since mimosine is a potent iron chelator for different types of cells but the inhibition of cyclin D1 expression is speci®cally found in H226 cells but not in H322 and H358 cells, it is doubtful that the iron deprivation activity of mimosine is the major cause that leads to cyclin D1 downregulation. Induction of p21 CIP1 gene expression by mimosine was previously reported in human 21PT breast cancer cells [24]. We also found that mimosine effectively increased the protein levels of p21 CIP1 in H226 and H358 cells. Furthermore, in accordance with the

Alpan's results, mimosine induced p21 CIP1 expression in a p53-independent manner. The effect of mimosine on the expression of p27 KIP1 and p57 KIP2, the other two members of the CIP/KIP gene family, has not been reported. Our results demonstrated that the expression of these two CDKIs was differentially regulated by mimosine in human lung cancer cell lines. Upregulation of p27 KIP1 by mimosine is an important ®nding because a recent study has clearly demonstrated that p27 KIP1 expression was signi®cantly reduced in nonsmall cell lung carcinomas and the authors found that decreased expression of p27 KIP1 was signi®cantly related to higher mortality [35]. Therefore, it is possible that mimosine may be a useful drug for the induction of p27 KIP1 in cancer cells and for the treatment of human non-small cell lung cancer. Potential involvement of p57 KIP2 in the development of lung cancer has been recently suggested [36]. However, the expression of p57 KIP2 is not controlled by mimosine. A previous study has demonstrated that inhibition of DNA synthesis in exponentially proliferating cells began 30±60 min after mimosine addition and was completed after 2±3 h [6]. However, alterations of cyclin D1 and CDKI expression induced by this drug were observed at 12± 24 h after addition and these results suggest that the mimosine-triggered change of cell cycle regulatory proteins may represent a sequential effect of blockage of DNA synthesis. Although mimosine does not affect cyclin D1 and CDKI gene transcription directly, changes in the expression of these cell cycle regulatory proteins may contribute to long-term growth arrest induced by mimosine. More functional assays must be performed to answer this question. Although originally considered as a late G1 blocker, it becomes clear that mimosine has multiple targets in vivo. The apparent inhibitory effect of mimosine on cyclin D1 expression found in this work suggests that this drug may block cell cycle progression in earlier G1 phase. Another study indicated that mimosine inhibited cyclin E-associated kinase activity and blocked cell growth in late G1 phase [24]. Moreover, this drug has also been found to prevent the serum-stimulated synthesis of cyclin A proteins and activation of cyclin A-associated histone H1 kinase activity which put mimosine in the category of S phase inhibitors [37]. Taken together, it is concluded that mimosine may potently inhibit lung cancer cell proliferation and may affect the cell

H.C. Chang et al. / Cancer Letters 145 (1999) 1±8

cycle progression at multiple points by regulating the expression of cyclins and the activity of CDKs. Indeed, our preliminary results indicate that mimosine exerts a signi®cant anti-cancer effect in nude mice (data not shown). However, the reason why mimosine can differentially control the expression of cyclins and CDKIs in different cancer cell lines remains unknown. The elucidation of these mechanisms will be helpful for the understanding of the action of mimosine and for the application of this drug in the therapy of lung cancer. Acknowledgements This work was supported by the grant CCMP-87RD-059 from the Committee on Chinese Medicine and Pharmacy, Department of Health, Taiwan ROC. References [1] M. Lalande, A reversible arrest point in the late G1 phase of the mammalian cell cycle, Exp. Cell Res. 186 (1990) 332± 339. [2] P.J. Mosca, P.A. Dijkwel, J.L. Hamlin, The plant amino acid mimosine may inhibit initiation at origins of replication in Chinese hamster cells, Mol. Cell Biol. 12 (1992) 4375±4383. [3] Y. Wang, J. Zhao, J. Clapper, L.D. Martin, C. Du, E.R. DeVore, K. Harkins, D.L. Dobbs, R.M. Benbow, Mimosine differentially inhibits DNA replication and cell cycle progression in somatic cells compared to embryonic cells of Xenopus laevis, Exp. Cell Res. 217 (1995) 84±91. [4] T.A. Hughes, P.R. Cook, Mimosine arrests the cell cycle after cells enter S-phase, Exp. Cell Res. 222 (1996) 275±280. [5] Y. Dai, B. Gold, J.K. Vishwanatha, S. Rhode, Mimosine inhibits viral DNA synthesis through ribonucleotide reductase, Virology 205 (1994) 210±216. [6] D.M. Gilbert, A. Neilson, H. Miyazawa, M.L. DePamphelis, W.C. Burhans, Mimosine arrests DNA synthesis at replication forks by inhibiting deoxynucleotide metabolism, J. Biol. Chem. 270 (1995) 9597±9606. [7] H. Lin, R. Falchetto, P.J. Mosca, J. Shabanowitz, D.F. Hunt, J.L. Hamlin, Mimosine targets serine methyltransferase, J. Biol. Chem. 271 (1996) 2548±2556. [8] S. Nyholm, G.J. Mann, A.G. Johansson, R.J. Bergeron, A. Graslund, L. Thelander, Role of ribonucleoide reductase in inhibition of mammalian cell growth by potent iron chelators, J. Biol. Chem. 268 (1993) 26200±26205. [9] H.M. Hanauske-Abl, M.H. Park, A.R. Hanauske, A.M. Popowitcz, M. Lalande, J.E. Folk, Inhibition of the G1/S transition of the cell cycle by inhibitors of deoxyhypusine hydroxylation, Biochim. Biophys. Acta 1221 (1994) 115±124. [10] D.J. Lew, S. Kornbluth, Regulatory roles of cyclin dependent

[11] [12] [13] [14] [15]

7

kinase phosphorylation in cell cycle control, Curr. Opin. Cell Biol. 8 (1996) 795±804. C.J. Sherr, Mammalian G1 cyclins, Cell 73 (1993) 1059± 1065. T. Hunter, J. Pines, Cyclins and cancer, Cell 66 (1991) 1071± 1074. J. Pines, Cyclins and cyclin-dependent kinase: take your partners, Trends Biochem. Sci. 18 (1993) 195±197. S. van den Heuvel, E. Harlow, Distinct roles for cyclin-dependent kinases in cell cycle control, Science 262 (1993) 2050± 2054. A. Amon, M. Tyers, B. Futher, B. Nsmyth, Mechanisms that help the yeast cell cycle clock tick: G2 cyclins transcriptionally activate G2 cyclins and repress G1 cyclins, Cell 74 (1993) 993±1007.

[16] T.M. Guadagno, J.W. Newport, Cdk2 kinase is required for entry into mitosis as a positive regulator for Cdc-cyclin B kinase activity, Cell 84 (1996) 73±82. [17] C.J. Sherr, J.M. Roberts, Inhibitors of mammalian G1 cyclindependent kinases, Genes Dev. 9 (1995) 1149±1163. [18] M. Serrano, G.J. Hannon, D. Beach, new regulatory motif in cell cycle control causing speci®c inhibition of cyclin D/ CDK4, Nature 366 (1993) 704±707. [19] H. Hirai, M.F. Roussel, J.Y. Kato, R.A. Ashmun, C.J. Sherr, Novel INK4 proteins, p19 and p18, are speci®c inhibitors of the cyclin D-dependent kinases CDK4 and CDK6, Mol. Cell Biol. 15 (1995) 2672±2681. [20] W. El-Deiry, T. Tokino, V.E. Velculescu, D.B. Levy, R. Parsons, J.M. Trent, D. Lin, W.E. Mercer, K.W. Kinzler, B. Vogelstein, WAF1, a potential mediator of p53 tumor suppression, Cell 75 (1993) 817±825. [21] K. Polyak, M.H. Lee, H. Erdjument-Bromage, A. Koff, J.M. Roberts, P. Tempst, J. Massague, Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals, Cell 78 (1994) 59±66. [22] C.J. Sherr, phase progression: cycling on cue, Cell 79 (1994) 551±555. [23] J. Massaque, K. Polyak, Mammalian antiproliferative signals and their targets, Curr. Opin. Genet. Dev. 5 (1995) 91±96. [24] R.S. Alpan, A.B. Pardee, p21WAF1/CIP1/SDI1 is elevated through a p53-indenpendent pathway by mimosine, Cell Growth and Differ. 7 (1996) 893±901. [25] W.C. Hung, J.S. Huang, L.Y. Chuang, Antisense oligodeoxynucleotides targets against different regions of cyclin D1 mRNA may exert different inhibitory effects on cell growth and gene expression, Biochem. Biophys. Res. Commun. 220 (1996) 719±723. [26] T. Mitsudomi, S.M. Steinberg, M.M. Nau, D. Carbone, D. D'Amico, S. Bodner, H.K. Oie, R.I. Linnoila, J.L. Mulshine, J.D. Minna, A.F. Gazdar, p53 Gene mutations in non-smallcell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features, Oncogene (1992) 171±180. [27] M.E. Ewen, H.K. Sluss, C.J. Sherr, H. Matsushime, J.Y. Kato, D.M. Livingston, Functional interactions of the retinoblastoma protein with mammalian D-type cyclins, Cell 73 (1993) 487±497.

8

H.C. Chang et al. / Cancer Letters 145 (1999) 1±8

[28] H. Matsushime, M.E. Ewen, D.K. Strom, J.Y. Kato, S.K. Hanks, M.F. Roussel, C.J. Sherr, Identi®cation and properties of an atypical catalytic subunit (p34/cdk4) for mammalian D type G1 cyclins, Cell 71 (1992) 323±334. [29] T. Motokura, T. Bloom, H.G. Kim, H. Juppner, J. Ruderman, H.M. Kronenberg, A. Arnold, A novel cyclin encoded by a bcl-linked candidate oncogene, Nature 350 (1991) 512±515. [30] E. Schurring, E. Verhoeven, W.J. Mooi, R.J.A.M. Michalides, Identi®cation and cloning of two overexpressed genes U21B31/ PRAD1 and EMS1, within the ampli®ed chromosome 11q13 region in human carcinoma, Oncogene 7 (1992) 355±361. [31] W. Jiang, S.M. Kahn, N. Tomita, Y.L. Zhang, S.H. Lu, I.B. Weinstein, Ampli®cation and expression of the human cyclin D gene in esophageal cancer, Cancer Res. 52 (1992) 2980± 2983. [32] B. Herber, M. Truss, M. Beato, R. Muller, Inducible regulatory elements in the human cyclin D1 promoter, Oncogene 9 (1994) 1295±1304. [33] J.N. Navoie, G. L'Allemain, A. Brunt, R. Muller, J. Pouysse-

[34]

[35]

[36]

[37]

gur, Cyclin D1 expression is regulated positively by the p42/ p44MAPK and negatively by the p38/HOGMAPK pathway, J. Biol. Chem. 271 (1996) 20608±20616. K.S. Kulp, S.L. Green, P.R. Vulliet, Iron deprivation inhibits cyclin-dependent kinase activity and decreases cyclin D/ CDK4 protein levels in asynchronous MDA-MB-453 human breast cancer cells, Exp. Cell Res. 229 (1996) 60±68. Y. Yatabe, A. Masuda, T. Koshikawa, S. Nakamura, T. Kuroishi, H. Osada, T. Tahashi, T. Mitsudomi, T. Takahashi, p27KIP1 in human lung cancers: differential changes in small cell and non-small cell carcinomas, Cancer Res. 58 (1998) 1042±1047. M. Kondo, S. Matsuoka, K. Uchida, H. Osada, M. Nagatake, K. Takagi, J.W. Harper, T. Takahashi, S.J. Elledge, T. Takahashi, Selective maternal-allele loss in human lung cancers of the maternally expressed p57KIP2 gene at 11p15.5. Oncogene 12 (1996) 1365±1368. S.T. Feldman, A. Schothal, Negative regulation of histone H1 kinase expression by mimosine, a plant amino acid, Cancer Res. 54 (1994) 494±498.