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Selective Sensitization of Retinoblastoma Protein-deficient Sarcoma Cells to Doxorubicin by Flavopiridol-mediated Inhibition of Cyclin-dependent Kinase 2 Kinase Activity¹

Laboratory of Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

	ABSTRACT

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We examined the effects of flavopiridol (FP), a cyclin-dependent kinase inhibitor, on doxorubicin (DOX)-induced cell killing in an osteosarcoma cell line (SaOs-2) that lacks functional retinoblastoma protein (pRb). The IC₅₀ value for DOX was 7-fold lower when combined with a low dose (100 nM) FP in pRb-deficient SaOs-2 cells than in the absence of FP. In contrast, the IC₅₀ value for DOX was not decreased in the presence of 100 nM FP in pRb-restored SaOs-2 cells. Consistent with this, FP enhanced DOX-induced activation of caspase-3, which correlates with apoptosis, in pRb-deficient cells but not in pRb-restored cells. Additional studies showed that FP decreased DOX-induced cell accumulation in S phase in retinoblastoma-restored cells but not in pRb-deficient cells. An increased expression of p21 and inhibition of cyclin-dependent kinase 2 kinase activity by FP was also observed in pRb-deficient cells but not in retinoblastoma-restored SaOs-2 cells. We conclude that pRb plays a key role in determining whether FP selectively sensitizes DOX-induced cell killing in human sarcoma cells. Because lack of functional pRb is a common abnormality in human cancers, the combination of FP with DOX in tumors lacking pRb would be worthy of further investigation.

	INTRODUCTION

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Sensitivity of human tumor cells to various DNA-damaging agents, including anticancer drugs, depends greatly on cell cycle checkpoint function (1) . The G₁-S checkpoint is regulated by the cyclin D1/cdk4,6⁴ and cyclin E/cdk2 complexes, which target the pRb and inactivate this protein by phosphorylation (2) . pRb is a positive regulator that binds to members of the E2F transcriptional factor family (E2F-1–3) in its hypophosphorylated form and inhibits E2F-1-mediated G₁-S progression (3) . An absence of pRb or phosphorylation of this protein by cyclin D1/cdk4,6 and cyclin E/cdk2 leads to release of functional E2F-1 and increases transition from G₁ into S phase. However, in S phase, E2F-1 is phosphorylated by cyclin A/cdk2 complex, resulting in loss of its DNA-binding activity and transactivation function (4) . Thus, in the presence of pRb, inhibition of cyclin D1/cdk4,6 or cyclin E/cdk2 by cyclin/cdk inhibitors such as p21 will reduce release of E2F-1 and dominantly arrest cells at G₁. In the absence of pRb, inhibition of cyclin A/cdk2 by cyclin/cdk inhibitors would increase unphosphorylated E2F-1, which may lengthen the S phase or block cells in the S-G₂ stage and enhance sensitivity of cells to S-phase-specific drugs such as DOX (5) .

FP, a flavone derivative with potent antitumor activity, has been demonstrated to be a potent cdk inhibitor (6 , 7) . FP inhibits activity of cdk2 and cdk4 by binding to the ATP-binding site of these cdks and causes a G₁ block (8) . FP may also directly inhibit cyclin B1/cdc2 kinase activity and result in induction of G₂ arrest (9) . Several studies (10 , 11) indicate that at higher concentrations, FP could inhibit other tyrosine protein kinases and decrease expression of cyclin D1 and bcl-2. Therefore, FP may play an important role in mediating cellular response to anticancer drugs by regulating cell cycle checkpoints.

In this study, we tested the hypothesis that in pRb-positive cells, FP may mainly arrest cells in G₁ and decrease the cellular response to S-phase-specific drugs. In contrast, in cells that lack functional pRb, FP may dominantly inhibit cyclin A/cdk2 activity, thus increasing functional E2F-1 activity and resulting in S-G₂ arrest. As a consequence, cells lacking pRb will be more sensitive to S-phase-specific drugs. We found that FP selectively sensitizes pRb-deficient SaOs-2 cells but not pRb-reconstituted SaOs-2 cells to DOX and show that this may be attributed to a FP-mediated S-G₂ block because of inhibition of cdk2 kinase activity.

	MATERIALS AND METHODS

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Chemicals.
FP, kindly provided by Dr. Edward Sausville (National Cancer Institute, Bethesda, MD), was dissolved in DMSO as a 2 mM stock solution. DOX was obtained from Sigma Chemical Co. [-³²P]ATP was from DuPont New England Nuclear (Boston, MA). Polyclonal antibodies to p21, cyclin A, cdk2, bcl-2, bcl-X/L, and E2F-1 and monoclonal antibodies to cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Lines and Cell Culture.
SaOs-2, an osteosarcoma cell line that lacks both p53 and functional pRb (12 , 13) , was obtained from the American Type Culture Collection. SaOs-2/9B and SaOs-2/10B are Rb-restored SaOs-2 sublines that were established by transfecting the plasmid pLRBRNL containing Rb cDNA into SaOs-2 cells with the transfection reagent N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate (14) . SaOs-2^neo is a G-418-resistant but pRb-deficient subline. All of the cell lines were maintained as monolayer cultures in RPMI 1640 containing 10% fetal bovine serum.

Cytotoxicity Assay.
Cytotoxicity to drugs was determined by the SRB cytotoxicity assay carried out in 96-well microtiter plates as described previously (15) . Cells were plated in duplicate wells (5000 cells/well) and exposed to DOX or DOX plus FP at different concentrations. Cells were fixed with 50% TCA solution for 1 h, and 0.4% SRB (Sigma Chemical Co.) was added to each well. After a 30-min incubation, the plates were washed with 1% acetic acid and read at 570 nm on a Biowhitaker microplate reader 2001. The wells with cells containing no drugs and with medium plus drugs but no cells were used as positive and negative controls, respectively.

Determination of Caspase-3 Activation.
A modified method described by Wang et al. (16) was used to examine caspase-3 activation. Drug-treated and untreated cells were lysed in hypotonic buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 100 µM phenylmethylsulfonyl fluoride, aprotonin (2 µg/ml), pepstatin (2 µg/ml), and leupeptin (2 µg/ml). The supernatants were collected and incubated with 100 µM DEVD-PNA as substrate at 37°C. The reaction was measured by change in absorbance at 405 nm using a plate reader.

Cell Cycle Analysis.
Exponentially growing cells were untreated or treated with drugs for 24 h. Cells were then collected and fixed with ice-cold 70% methanol. DNA was stained with propidium iodide (Calbiochem, San Diego, CA) as described previously (17) . Ten thousand stained cells were analyzed on a Becton Dickinson fluorescence-activated cell sorter.

Western Blot Analysis.
Exponentially growing cells were treated with DOX or DOX plus FP for 24 h. Cell extracts (100 µg) were obtained and subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with various primary antibodies using standard techniques (18) . Protein was detected using enhanced chemiluminescence detection.

Immunoprecipitation and Histone-H1 Kinase Assay.
Cells were exposed to DOX and DOX plus FP for 24 h and were lysed with a solution containing: 100 mM Tris-HCl (pH 7.5), 300 mM NaCl, 2% NP-40, 0.5% sodium deoxycholate, 0.2% SDS, and protease inhibitors. After centrifugation at 4°C for 15 min, the supernatant was collected and precleared with protein A/G agarose for 30 min. Precleared protein extract (200 µg) was combined with lysis buffer and 10 µg of anti-cdk2/agarose conjugate (Santa Cruz Biotechnology) and incubated for 1 h at 4°C with shaking. Agarose beads were collected by centrifugation and washed four times in lysis buffer and once in kinase buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl₂, 2 mM EGTA, and 1 mM DTT). The beads were resuspended in 20 µl of kinase buffer containing 20 µM ATP, 100 mg/ml histone H1 (Boehringer Mannheim), and 200 µCi/ml [-³²P]ATP. After incubation for 20 min at 30°C, the reaction mixture was then subjected to 7.5% SDS-PAGE followed by autoradiography.

	RESULTS

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FP Increases DOX-induced Cell Killing and DOX-induced Activation of Caspase-3 in pRb-deficient Cells.
The sensitivity of parental SaOs-2 cells and two Rb-restored sublines, SaOs-2/9B and SaOs-2/10B, to FP was first determined. Cells were treated with FP at different concentrations for 5 days. As shown in Fig. 1A , sensitivity of SaOs-2/9B and 10B cell lines to FP was similar to that of SaOs-2^neo cells. IC₅₀s for these cell lines were approximately 500 nM. The sensitivity to DOX in these cell lines was measured next, and no obvious difference in sensitivity was observed between SaOs-2^neo cells and pRb-restored cells (Fig. 1B) . To determine whether FP would affect sensitivity of pRb-deficient or -restored cells to DOX, the effect of DOX combined with FP (100 nM), a dose that caused less than 10% inhibition of cell growth, was examined on these cell lines. As shown in Fig. 1B , the effect of DOX on SaOs-2 cells was greatly enhanced by the addition of this concentration of FP. IC₅₀s for DOX combined with FP were 7-fold lower compared with that for DOX alone (2.6 nM versus 18.5 nM) in Rb-deficient cells. In contrast to this result, IC₅₀s for DOX combined with FP were similar to that for DOX alone in Rb-restored cells. To understand whether FP enhancement of DOX-induced cell killing links to increased apoptotic response to DOX in pRb-deficient cells, we further determined caspase-3 activation, which correlates with apoptosis (16) . As shown in Fig. 2 , FP alone did not increase caspase-3 activity in either pRb-deficient cells or pRb-restored cells. DOX caused a moderate increase in caspase-3 activity in both pRb-deficient and -restored cells. However, when FP was combined with DOX, substantially enhanced DOX-induced activation of caspase-3 (about 2-fold higher than that for DOX alone) was observed after either 4-h or 24-h exposure to drugs in pRb-deficient cells but not in pRb-restored cells.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Effect of FP on DOX-induced cell killing in human sarcoma cells. Cells were treated with FP alone, DOX alone, or FP combined with DOX for 5 days. Assays were carried out with the use of the SRB assay. A, FP alone; B, DOX alone and combined with FP (100 nM). , SaOs-2neo cells; •, SaOs-2/9B; , SaOs-2/10B; , FP; ----, DOX; ····, FP + DOX. The results are representative of three individual experiments.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Effect of FP on DOX processing of caspase-3 in pRb-deficient and -restored SaOs-2 cells. Cells were exposed to FP alone, DOX alone, or FP combined with DOX for 4 h or 24 h. Caspase-3 activity was measured as described in "Materials and Methods." A, SaOs-2neo; B, SaOs-2/9B; , control; , FP (100 nM); , DOX (500 nM); , DOX (500 nM) + FP (100 nM). The results represent the average value from two independent experiments.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Effect of FP on cell cycle distribution in pRb-deficient and -restored SaOs-2 cells. Cells were treated with FP (100 nM), DOX (500 nM), or FP (100 nM) combined with DOX (500 nM), respectively, for 24 h. Then cells were collected, fixed with 70% methanol, and stained with propidium iodide. 10,000 stained cells were then analyzed for each sample by flow cytometric analysis.

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Immunoblot analysis of cyclin D1, cyclin A, cdk2, E2F-1, p21, and bcl-X/L after cells were treated with FP, DOX, or FP combined with DOX. Cells were exposed to drugs for 24 h, whole cell lysates were extracted, and expression of various proteins was determined by Western blot analysis. Lanes 1 and 5, control (without drug treatment); Lanes 2 and 6, DOX (500 nM); Lanes 3 and 7, FP (100 nM); Lanes 4 and 8, FP(100 nM) + DOX(500 nM).

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Effect of FP on cdk2 kinase activity in human sarcoma cells. Cells were exposed to FP, DOX, and FP combined with DOX, respectively, for 24 h, and the total cellular extract (containing 200 µg of protein) was subjected to immunoprecipitation with anti-cdk2 antibody-agarose conjugates followed by histone H1 assay as described in "Materials and Methods." Lanes 1 and 5, control (without drug treatment); Lanes 2 and 6, FP (100 nM); Lanes 3 and 7, DOX (500 nM); Lanes 4 and 8, FP(100 nM) + DOX (500 nM).

	DISCUSSION

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In this study, we demonstrated that FP selectively enhances the cytotoxicity of DOX in Rb-deficient sarcoma cells. Although FP was reported to have both functions of inducing apoptosis and arresting cell cycle, enhancement of DOX effect by FP in this study is more likely through its cell cycle arrest rather than apoptosis because the concentration of FP used here is subtoxic and different cell cycle distributions generated by FP were observed between Rb-deficient and -restored cells. By measuring the levels of cell cycle-regulatory proteins and associated kinase activity, additional results indicated that FP-modulated DOX sensitivity in Rb-deficient SaOs-2 cells may involve preferable inhibition of cdk2 activity. Inhibition of cyclin A/cdk2 activity would reduce phosphorylation of E2F-1, which results in an increase of cells in late S phase and, as a consequence, increases susceptibility of cells to DOX- induced apoptosis as shown previously (5) and in this study. Therefore, a pathway that links FP enhancement of DOX sensitivity is inhibition of S-phase cyclin/cdks by FP and pRb status of the cell.

Several studies have demonstrated that, in the absence of pRb, no or little cdk4 activity is detected, and cyclin D1 is dispensable (6 , 22) . As a consequence, the regulatory interplay between cdk4, D-type cyclins, p16, and pRb may be lost (23) . Therefore, in the absence of pRb, interaction of FP with cdk4 may be attenuated, and cdk2 would become a major target of FP. Thus, these studies also seem to support the possibility that FP preferably targets S-phase cdks in pRb-deficient cells.

In the presence of pRb, FP does not induce p21 expression or even inhibit DNA damaging agents-induced p21 expression (20) . We also observed a lack of induced p21 expression by FP in pRb-positive cells. However, we did observe that FP increased p21 expression in pRb-deficient cells. Increased expression of p21 in the absence of pRb has been demonstrated to increase the S-phase fraction of human sarcoma cells and enhance sensitivity of these cells to anticancer drugs (5 , 24) . Therefore, increased p21 expression by FP may partly contribute to enhancement of DOX sensitivity in pRb-deficient cells. It is not clear how FP increases p21 expression in the absence of pRb. However, several studies showed that E2F-1 is able to induce p53-independent p21 expression by directly transactivating the p21 promoter (25) . Because "free" E2F-1 is much higher in pRb-deficient cells than in pRb-positive cells (14) and FP may further increase activity of E2F-1 by inhibition of cdk2, the FP-induced increase in p21 expression in the absence of pRb may be through the E2F-1-p21 pathway.

Although DOX is one of the most active drugs to treat human sarcomas, its effectiveness against this disease is limited. Because loss of functional pRb occurs frequently in sarcomas (26) , administration of FP combined with DOX to selectively enhance the DOX sensitivity in pRb-deficient cells may improve the antitumor effects of DOX in patients with sarcomas. This combination is worthy of further exploration in this disease and in other pRb-deficient tumors.

	FOOTNOTES

 
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 Supported by a USPHS Grant # PO1-CA-47179.

² To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology and Therapeutics, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-8230; Fax: (212) 639-2767; E-mail: bertinoj{at}MSKCC.org

³ American Cancer Society Professor of Medicine and Pharmacology.

⁴ The abbreviations used are: cdk, cyclin-dependent kinase; pRb, retinoblastoma protein; FP, flavopiridol; DOX, doxorubicin.

Received 6/30/00. Accepted 1/16/01.

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