[CANCER RESEARCH 58. 704-710. February 15. I998| Inhibition of Cyclin Dl Expression and Phosphorylation of Retinoblastoma Protein by Phosmidosine, a Nucleotide Antibiotic1 Hideaki Kakeya, Rie Onose, Phillip C.-C. Liu,2 Chizuko Onozawa, Fuini«Matsumura, and Hiroyuki Osada3 Antibiotics Laboratory, The Insiliate of Physical ami Chemical Research (RIKEN), Saitama 35I-OI9N, Japan ¡H.K., R. O., C. O.. H. O.¡,and Department of Environmental Toxicology, and Institute of Toxicology and Environmental Health. University of California. Davis. California 95695 {P. C.-C. L., F. M.J ABSTRACT implicated in the phosphorylation of pRB. Ectopie expression of cyclin Dl induces phosphorylation of pRB and accelerates progres In this report, we studied the effect of phosmidosine, a proline-contain- sion through G, (14, 15). In addition, microinjection of either anti- ing nucleotide on the serum-induced cell cycle progression in human lung fihroblast YVI-38 cells. Phosmidosine suppressed S-phase entry and ar cyclin Dl antibodies or antisense plasmid prevents cells from entering rested cell cycle progression at the I., phase. In serum-stimulated cells, S phase (14, 16, 17). phosmidosine did not affect the activation of the mitogen-activated protein Thus, understanding the underlying biochemical processes on the kinase cascade. However, phosmidosine inhibited hyperphosphorylation pRB functions will advance our knowledge of cellular proliferation in of retinohlastoma (RB) protein by RB-kinases such as cyclin-dependent general and also will have implications for efficient cancer therapies. kinase 4 and cyclin-dependent kinase 2, probably as a result of the Compounds that arrest cells at a specific point in the cell cycle have inhibition of cyclin Dl expression. Furthermore, in tsFT210 cells, a tem proven to be extremely useful tools for unraveling cell cycle regula perature-sensitive <•</<•_'mutantisolated from the mouse mammary carci tory events and might be useful for cancer chemotherapy. noma cell line FM3A, phosmidosine, irreversibly inhibited the cell cycle Phosmidosine (Fig. 1) inhibited the cell cycle progression at G, progression at G, without affecting the (., to M transition. Phosmidosine with the flat morphological reversion activity in NRK cells trans acts at an earlier point in G, compared with mimosine or aphidicolin, formed by temperature-sensitive Rous sarcoma virus (18-20). Its well-known cell cycle blockers at the G,-S boundary. Taken together, phosmidosine arrested cells at a spirille point between the start point and mechanism of action, however, has remained elusive. In this report, restriction point in <;, and is a useful drug that may contribute to the we show that phosmidosine treatment of a mouse carcinoma cell line understanding of the regulatory mechanisms of G, progression. as well as a human normal cell line arrested cell cycle progression at G,. Concomitantly, phosphorylation of pRB and expression of cyclin Dl are blocked. These effects are irreversible because cells from INTRODUCTION which phosmidosine has been washed out remain arrested at G,. The RB4 gene is an antioncogene, the inactivation of which is often Because phosphorylation of pRB is essential for the G,-to-S transi tion, our experiments suggest that phosmidosine blocks cell growth by observed in various kinds of tumors including RBs and osteosarcomas ( 1). The RB gene product (pRB) is a nuclear protein of Mr 110,000- inhibiting hyperphosphorylation of pRB through the suppression of the cyclin Dl expression. 115.(XX)that forms complexes with the transforming protein encoded by DNA tumor virus, such as the adenovirus EIA protein, the SV40 (SV40) large T antigen, or the papilloma virus E7 protein. Recent MATERIALS AND METHODS genetic studies have revealed that complex formation between pRB and oncoproteins is closely linked with viral transforming activities Materials. Phosmidosine was isolated from Streptomyces durhameusis as (2-4). pRB also interacts specifically with several cellular proteins, described previously (18, 20). Human lung fibroblast WI-38 cells were ob including the transcription factor E2F and the proto-oncogene product tained from the RIKEN Cell Bank (Tsukuba, Japan). tsFT210 cells, a temper ature-sensitive cdc2 mutant isolated from the mouse mammary carcinoma cell Myc (5. 6). It is well known that pRB is phosphorylated in a cell cycle-dependent manner: hypophosphorylated in the G,,-G, phase and line FM3A, were a kind gift from Dr. F. Hanaoka (RIKEN; Ref. 21). MBP and hyperphosphorylated in the G,-to-S transition. Phosphorylation of histone HI were purchased from Sigma Chemical Co. (St. Louis, MO); and anti-cyclin Dl, anti-CDK2, and anti-CDK4 antibodies were purchased from pRB results in the dissociation of pRB and transcription factor, which Upstate Biotechnology (Lake Placid, NY). Anti-pRB antibody (PMG-245) was induces the S-phase entry (5, 7-9). obtained from PharMingen (San Diego, CA), and [7-32P]ATP was from 1CN Concomitant with these findings, pRB was shown to be phospho Biochemicals, Inc. (Costa Mesa, CA). rylated by CDKs in vitro and in vivo (10-13). CDK2/cyclin E com Cell Synchronization. WI-38 cells were grown at 37°Cin 5% CO2 in plex as well as CDK4/cyclin D phosphorylate pRB. Recent analysis of DMEM containing 10% FCS. Exponentially growing WI-38 cells were ar phosphorylation sites both in vitro and in vivo indicated that pRB is rested at G0 by incubation in DMEM medium supplemented with 0.2% PCS phosphorylated on more than 10 serine or threonine residues but is not for 3 days. Then these quiescent cells were stimulated by the addition of 10% phosphorylated on tyrosine residue. Cyclins of the D class (Dl, D2, FCS. tsFT2IO cells were maintained in RPMI 1640 with 10% PCS at the per and D3) complexed with CDK.4 or CDK6 are most prominently missive temperature of 32°Cand cultured at the nonpermissive temperature of 39°Cfor 17 h to synchronize them at the G2-M boundary. They were incubated Received 9/3/97; accepted 12/16/97. at 32°Cfor 4 h to induce their entry into G, (22). The costs of publication of this article were defrayed in part by the payment of page Flow Cytometric Analysis. Flow cytometric analysis was performed es charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sentially as described by Noguchi and Browne (23). The harvested cells were 1This work was supported in part by a Special Grant for Promotion of Research stained with solution containing 50 fig/ml propidium iodide. 0.1% sodium (RIKEN). a Grant for Multibioprobes (RIKEN), and a Gram from the Ministry of citrate, and 0.2% NP40 and analyzed for DNA contents using a flow cytometer Education. Science. Sports and Culture. Japan. - Present address: Department of Biological Chemistry, University of Michigan Med (Profile II: Coulter Co., Hialeah, FL). [3H]Thymidine Incorporation. WI-38 cells were plated in 48-well plates ical School. Ann Arbor. Ml 48109-0606. ' To whom requests for reprints should be addressed, at The Institute of Physical and at 3 X IO4 cells/200 /il/well and cultured for 3 days. Cells were washed twice Chemical Research. RIKEN. Hirosawa 2-1. Wako-shi, Saitama .151-01, Japan. Phone: with serum-depleted DMEM and were incubated in DMEM containing 0.2% 81-48-467-9541; Fax: 81-48-462-4669; E-mail: [email protected]. 4 The abbreviations used are: RB. retinoblastoma; pRB. RB protein; CDK. cyclin- PCS at 37°Cfor 3 days. These quiescent cells were pretreated with various dependent kinase; MAP kinase, mitogen-activated protein kinase: MBP. myelin basic concentrations of phosmidosine for 15 min and stimulated with 10% FCS. protein: BrdUrd. 5-bromo-2'-deoxyuridine; CAK. CDK-activating kinase. After 18 h incubation in media with 10% FCS. the cells were labeled for 2 h 704 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1998 American Association for Cancer Research. PHOSMIDOSINE EFFECTS ON CYCLIN Dl AND pRB NH2 RESULTS Phosmidosine Arrests WI-38 Cell Cycle Progression at G,. The cell cycle progression of human lung fibroblast WI-38 cells was monitored by flow cytometry. Exponentially growing WI-38 cells had two peaks: 2 C DNA (G0-G, phase) and 4 C DNA (G2-M phase). A saddle peak between 2 C and 4 C peak was S phase, which was confirmed by the incorporation of BrdUrd, a thymidine analogue, into replicating DNA (Fig. 2, A-a and B-a). Phosmidosine (10 /MM)appar ently decreased the saddle peak area between 2 C and 4 C and the incorporation of BrdUrd (Fig. 2, A-b and B-b). After washing out the phosmidosine and allowing the cells to recover for 24 h, the cell cycle profile did not change appreciably (Fig. 2, A-c and B-c) from cells that were not allowed to recover (Fig. 2, A-b and B-b). These data indicate that phosmidosine irreversibly blocks the progression of WI-38 cells in G,. This arrest was not due to toxicity of the drug, as determined Fig. l. Structure of phosmidosine. by trypan blue exclusion (data not shown). Because phosmidosine appeared to arrest cycling cells at G,, we examined its effect on S-phase entry by quiescent WI-38 cells. with 2 fiCi/ml ['HJthymidine (25 mCi/ml; Amersham International, Bucking [3H]Thymidine incorporation was detected 16 h after serum addi hamshire, England), washed twice with 5% trichloroacetic acid, and lysed with tion, peaked after 23 h, and then declined (data not shown). We l N NaOH. The radioactive content of the resulting trichloroacetic acid- pretreated quiescent WI-38 cells with several concentrations of insoluble fractions was measured with a liquid scintillation counter (TriCurve; phosmidosine for 15 min, stimulated the cells with serum, and Packard, Meriden, CT). added [3H]thymidine 18 h later.