Cyclin E overexpression enhances cytokine-mediated ... - Nature

Genes and Immunity (2003) 4, 336–342 & 2003 Nature Publishing Group All rights reserved 1466-4879/03 $25.00 www.nature.com/gene

Cyclin E overexpression enhances cytokine-mediated apoptosis in MCF7 breast cancer cells NK Dhillon1 and M Mudryj1,2 Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA 95616, USA; VA NCHS, Mather, CA 95655, USA

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Cyclin E, the regulatory component of the cyclin E/cyclin-dependent kinase (CDK) complex, is required for proliferation and overexpression of this cyclin is associated with many types of human tumors. To elucidate the mechanism by which cyclin E overexpression promotes tumorigenesis, cyclin E was overexpressed in two breast cancer lines: MCF7 and T47D. Cells overexpressing cyclin E display a marked decrease in the expression of Bcl-2, an antiapoptotic protein, and increased levels of the proapoptotic proteins Bad and Bax. The levels of Bcl-XL and Mcl-1 remain unchanged. Since the homeostasis of pro- and antiapoptotic proteins was altered, we asked if cyclin E overexpression modifies responses to cytokines. MCF7 cyclin E overexpressing cells have an enhanced sensitivity to Fas, TRAIL, and TNF-a-induced apoptosis. T47D cells overexpressing cyclin E have a significant increase in TNF-a and TRAIL-induced apoptosis. In conclusion, our results provide a link between expression of cyclin E, deregulation of Bcl-2, and an altered response to cytokine-mediated apoptosis. Genes and Immunity (2003) 4, 336–342. doi:10.1038/sj.gene.6363973 Keywords: Cyclin E; TRAIL; Bcl-2; apoptosis; breast cancer; cytokine

Introduction Progression through the cell cycle is regulated by cyclins and their catalytic subunits, the cyclin-dependent kinases (CDKs). Cyclin E, an essential cyclin that is expressed in late G1 and early S phase, regulates the phosphorylation of many proteins that are important for cell proliferation, including the retinoblastoma protein (RB),1 BRCA1,2 and NPAT.3 Cyclin E is overexpressed in approximately a quarter of all breast tumors 4 and studies have found that cyclin E overexpression and an increase in cyclin E/ CDK2 kinase activity correlates with a poor prognosis.5,6 A recently published study found that cyclin E overexpression in stage 1 breast cancer is a very accurate predictor of negative clinical outcome.7 None of the patients with low levels of cyclin E died of breast cancer by 5 years, while all patients with high levels of cyclin E died within this time period. Induction of apoptosis is thought to be instrumental in promoting tumor regression and the Bcl-2 family of pro- and antiapoptotic proteins augments apoptotic responses. A positive ratio between pro- and antiapoptotic proteins leads to cytochrome C release from the mitochondria, resulting in the execution of the death pathway.8 Overexpression of antiapoptotic proteins Bcl-2 and Bcl-XL9–12 and downregulation of proapoptotic factors, such as Fas,13 has been found in many primary tumors and tumor cell lines. Paradoxically, several Correspondence: Dr M Mudryj, Department of Microbiology and Immunology, 3149 Tupper Hall, University of California, Davis, CA 95616, USA. E-mail: [email protected] Received 21 October 2002; revised 13 December 2002; accepted 17 December 2002

studies found that a high level of Bcl-2 expression in breast tumors is a positive not negative prognostic indicator.14,15 The molecular basis of this correlation is unknown. Immune surveillance is important for the elimination of transformed or malignant cells and cytokines are important for this process.16 The role of cytokines in preventing the growth of transformed cells has been utilized in the administration of synthetic cytokines in anticancer therapies.17,18 The ability of transformed cells to escape immune surveillance results in tumor growth. Tumor necrosis factor-a(TNF-a), Fas, and TRAIL (TNFrelated apoptosis inducing ligand) are three related cytokines that interact with their respective receptors to induce apoptosis.19 Previous studies have shown that TNF-a has antitumor activity and that activation of the TNF receptor can cause apoptosis and regression of tumors.20,21 The TNF-a relative Fas also has implications in the development of tumors.22 The molecular events triggered by the binding of Fas ligand to the Fas receptor have been well characterized. Induction of the Fas receptor by its Fas ligand results in an activation of apoptotic events involving the Bcl-2 family of pro- and antiapoptotic proteins and caspases (cysteine aspartic acid proteases).8,23 Fas-dependent activation of caspase-8 24 conveys an apoptotic signal via a proteolytic cascade, leading to the degradation of cellular targets, DNA fragmentation, and cell membrane blebbing.8 TRAIL is a novel anticancer agent that is capable of inducing apoptosis in a variety of human tumor cells both in cell culture and in vivo.25–27 TRAIL promotes apoptosis by binding to the transmembrane receptors TRAIL-R1/DR4 and TRAIL-R2/DR5.28 Its cytotoxic activity is relatively selective to the human tumor cell

Cyclin E enhances cytokine-mediated apoptosis NK Dhillon and M Mudryj

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lines with not much of an effect on normal cells.26,27 The susceptibility of tumor cells to TRAIL and an apparent lack of activity in normal cells have led to a proposed use of TRAIL in cancer therapy. In MCF7 breast cancer cells, TRAIL-, Fas-, and TNF-a-induced apoptosis is inhibited by the overexpression of Bcl-2.29,30 In the present study, we have investigated the role of cyclin E overexpression in apoptosis. We show that an overexpression of cyclin E in MCF7 and T47D cells results in a deregulation of pro- and antiapoptotic proteins. The levels of Bcl-2 are decreased, while the levels of the proapoptotic proteins Bax and Bad are elevated. Furthermore, cells overexpressing cyclin E have an increased sensitivity to cytokine-mediated apoptosis, suggesting that cyclin E-dependent alterations in gene expression modulates the apoptotic response.

Results Cyclin E overexpression in MCF7 and T47D breast cancer cells MCF7 and T47D cells overexpressing cyclin E (MCF7CycE and T47D-CycE) were generated by transfection of the RcCMV-cyclin E plasmid into MCF7 and T47D cells. A total of 40 MCF7 clones and 30 T47D were selected and analyzed for the expression of cyclin E by Western immunoblot analysis. Two MCF7 clones, MCF7-CycE 1 and MCF7-CycE 3, and two T47D clones, T47D-CycE 4 and T47D-CycE 18, have the highest levels of cyclin E and were selected for further analysis. MCF7-RcCMV and T47D-RcCMV controls cells were generated by transfection of the RcCMV parent vector into MCF7 and T47D breast tumor cells. Three independent control clones of each cell line were analyzed and all have comparable levels of cyclin E expression (data not shown). The controls with the highest level of cyclin E were used as the representative control in all experiments. As shown in Figure 1a, MCF7-CycE 1 and MCF7CycE 3 cells have a 5.0- and 9.9-fold increase in cyclin E levels, respectively. T47D-CycE 4 and T47D-CycE 18 cells have a 3.6- and 5.2-fold increase, respectively, in cyclin E expression. All the cyclin E expressing clones have smaller molecular weight forms. These truncated forms have been previously described in cyclin E overexpressing cell lines and tumors, and are N-terminally processed variants that have a higher activity.31 The cyclin E overexpressing cells also have higher levels of cyclin E-associated kinase activity (Figure 1b). Bcl-2 protein levels are decreased in cyclin E overexpressing cells Initial studies on cell cycle distribution of control and cyclin E overexpressing cells using laser scanning cytometry (LSC) indicated that cells overexpressing cyclin E display an increase in apoptosis when treated with estrogen-depleted media or 10 mM tamoxifen (32 and NKDF unpublished data). This led us to investigate whether cyclin E alters expression levels of the Bcl-2 family of pro- and antiapoptotic proteins. Western immunoblot analyses indicated that cyclin E decreases the protein levels of Bcl-2 by 53.4-fold in MCF7-CycE 1 cells, 503-fold in MCF7-CycE 3 cells, and 2.3-fold in T47D-CycE 4. Bcl-2 levels in T47D-CycE 18 cells were 2.4fold higher than in control cells (Figure 1c and Table 1).

Figure 1 Western blot analysis. (a) A total of 30 mg of total protein was used for each Western blot analysis. MCF7-CycE 1, MCF7-CycE 3, T47D-CycE 4, and T47D-CycE 18 cells have elevated levels of cyclin E as compared to control cells. Both bands represent cyclin E as this protein exists in two forms in these cells. Actin was used as loading controls and was used to standardize each sample. Quantitation of the bands was determined using Image Quant v3.0. (b) MCF7 and T47D cells have a concomitant higher cyclin Eassociated kinase activity as compared to controls. Histone H1 was used as substrate. (c) There are reduced levels of Bcl-2 in MCF7CycE 1, MCF7-CycE 3, and T47D-CycE 4 cells as compared to control cells. There is a slight decrease in Bcl-XL and Mcl-1 levels only in T47D cyclin E overexpressing cells. Bad and Bax levels are increased in both cell lines overexpressing cyclin E. Actin served as a loading control for each experiment.

The levels of the antiapoptotic proteins Mcl-1 and Bcl-XL were unchanged in MCF7 cells and slightly decreased in T47D cycE overexpressing cells. We also examined the levels of two proapoptotic Bcl-2 family members, Bad and Bax. As shown in Figure 1c and Table 1, MCF7 and T47D cyclin E overexpressing cells have approximately Genes and Immunity


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Table 1 Fold differences in protein levels of Bcl-2 family members in response to cyclin E overexpression Bcl-2

Bcl-XL

Bad

Bax

Mcl-1

MCF7 CycE 1 MCF7 CycE 3

53.4 503.2

1.1 1.3

+2.0 +2.6

+2.4 +1.6

+1.1 1.0

T47D CycE 4 T47D CycE 18

2.3 +2.4

1.3 2.8

+2.0 +1.4

+2.9 +2.9

1.8 2.9

+ indicates fold increase; indicates fold decrease.

two-fold higher protein levels of Bad than control cells. The levels of Bax are also elevated by approximately two-fold in MCF7 cyclin E overexpressing cells. However, the levels of Bax are elevated by approximately three-fold in T47D cyclin E cells. Changes in Mcl-1 are observed only in the T47D cells where it is decreased by 1.8-fold in T47D-CycE 4 cells and 2.9-fold in T47D-CycE 18 cells. Therefore, cyclin E represses the expression of the antiapoptotic Bcl-2 protein and stimulates the expression of proapoptotic proteins in two different cell lines, thus altering the balance of pro- and antiapoptotic proteins.

Cyclin E enhances cytokine-mediated apoptosis Previous studies reported that overexpression of Bcl-2 in MCF7 cells inhibits Fas, TNF-a, and TRAIL-mediated apoptosis.29,30 Therefore, we reasoned that a decrease of Bcl-2 might sensitize cells to these cytokines. Cell death was detected in two ways. First, trypan blue (Figure 2) was used to measure cell viability. Secondly, a complementary TUNEL assay was used to detect chromatin fragmentation, a hallmark of apoptosis (Figure 3). In this assay, cells were counterstained with propidium iodide to visualize DNA and then, LSC was used to quantitate apoptosis (Figure 3). The results obtained using the two different assays were nearly identical. We quantitated the amount of apoptosis in control and cyclin E overexpressing cells treated with cytokines. TNF-a-mediated apoptosis was induced by the addition of 100 ng/ml of TNF-a for 24 h. As shown in Figures 2 and 3, both MCF7 and T47D cyclin E overexpressing cells display a significant increase in the levels of apoptosis. Statistical significance was calculated using Student’s t-test. MCF7CycE 1 and MCF7-CycE 3 cells also display a statistically significant increase in cell death compared to MCF7 control cells when treated with 250 ng/ml of anti-Fas antibodies and 10 mg/ml of cyclohexamide (Figures 2a and 3a). Similar treatment of T47D cells causes complete apoptosis; therefore, the amount of Fas was reduced to 125 ng/ml and cyclohexamide was omitted. Under these conditions, cyclin E overexpression in T47D cells does not further augment their sensitivity to Fas (Figures 2b and 3b). MCF7 and T47D cyclin E overexpressing cells also both undergo considerable apoptosis in response to 50 ng/ml TRAIL (Figures 2 and 3). Cyclin E overexpression does not alter the levels of the TRAIL receptors DR4 and DR5 (Figure 1d). These results indicate that cyclin E overexpression sensitizes MCF7 and T47D cells to cytokine-mediated apoptosis. Genes and Immunity

Figure 2 Cyclin E overexpressing cells show greater Fas- and TNFa-mediated apoptosis than control cells. (a) Percentage of cell death because of treatment with TNF-a (100 ng/ml), Fas (250 ng/ml Fas and 10 mg/ml cyclohexamide), and TRAIL (50 ng/ml) in MCF7RcCMV (black bars), MCF7-CycE 1 (white bars), and MCF7-CycE 3 (striped bars). (b) Percentage of cell death because of stimulation of the in T47D-RcCMV (black bars), T47D-CycE 4 (white bars), and T47D-CycE 18 (striped bars) cells treated with TNF-a (100 ng/ml), Fas (125 ng/ml), and TRAIL (50 ng/ml). Viability was assayed using the trypan blue exclusion method and percentages were calculated based on the ratio of blue-stained cells and the total number of cells. The figures represent an average of at least three experiments. MCF7-CycE 1 and MCF7-CycE 3 cells display a statistically significant increase in cell death compared to MCF7 control cells when treated with TNF-a (P ¼ 0.0157; P ¼ 0.030), 250 ng/ml anti-Fas antibodies and 10 mg/ml cyclohexamide (P ¼ 0.0023; P ¼ 0.0001), and TRAIL (P ¼ 0.0168; P ¼ 0.0011). T47D-CycE 4 and T47D-CycE 18 displayed a statistically significant increase in cell death compared to T47D control cells when treated with TNF-a (P ¼ 0.0148; P ¼ 0.0143) and TRAIL (P ¼ 0.0109; P ¼ 0.0252).

Discussion The ratio of pro- and antiapoptotic proteins is thought to be an important factor in the execution of the programmed cell death pathway. Several studies have shown that overexpression of Bcl-2 in MCF7 breast cancer cells inhibits apoptosis induced by cytokines 29,30 as well as chemotherapeutic agents.13 In the current study, we used two breast cancer cell lines, T47D and MCF7, to study the role of cyclin E in apoptosis. Both cell lines are estrogen dependent and undergo arrest in response to the antiestrogen drug Tamoxifen.33 Both cell lines also have wild-type RB protein, but while MCF7 cells have wild-type p53, T47D cells harbor a mutant form of p53.34 In addition, MCF7 cells do not express caspase 3.35 MCF7 and T47D cells that overexpress cyclin E have deregulated cell cycle controls that promote growth even in the presence of Tamoxifen (32 and NKDFunpublished data). This current study argues


Figure 3 MCF7 and T47D cyclin E overexpressing cells have a greater apoptotic response to cytokines than control cells as verified by the TUNEL assay. (a) Graphic representation of TUNEL assay results obtained for MCF7 control and cyclin E overexpressing cells. Cells were treated as described previously with TNF-a, Fas, or TRAIL. (b) Graphic representation of TUNEL assay results obtained for T47D control and cyclin E overexpressing cells. All cells were exposed to fluorescein-12-dUTP, which results in green fluorescence in the nucleus of apoptotic cells only, and propidium iodide, which stains both apoptotic and nonapoptotic cells red. Apoptosis was quantified by measuring the amount of green fluorescence to the amount of total red fluorescence using a laser scanning cytometer. MCF7-CycE 1 and MCF7-CycE 3 cells display a statistically significant increase in apoptosis compared to MCF7 control cells when treated with TNF-a (P ¼ 0.0340; P ¼ 0.0062), 250 ng/ml antiFas antibodies and 10 mg/ml cyclohexamide (P ¼ 0.0130; P ¼ 0.0110), and TRAIL (P ¼ 0.0001; P ¼ 0.0045). T47D-CycE 4 and T47D-CycE 18 displayed a statistically significant increase in apoptosis compared to T47D control cells when treated with TNF-a (P ¼ 0.0105; P ¼ 0.0479) and TRAIL (P ¼ 0.006; P ¼ 0.0326).

that cyclin E overexpression also deregulates Bcl-2 and modifies the balance of pro- and antiapoptotic proteins. These results are particularly intriguing, since a recently published study noted a statistically significant correlation between cyclin E overexpression and decreased levels of Bcl-2 in breast tumors.35 Overexpression of cyclin E in MCF7 cells results in a precipitous drop of Bcl-2, which is due to a decrease in mRNA levels (NKDFunpublished data). T47D cells have very low, but detectable levels of Bcl-2. Overexpression of moderate levels of cyclin E (T47D-cycE 4) reduces the level of Bcl-2. However, a further increase in cyclin E (T47DCycE 18) results in increased amounts of Bcl-2 in comparison with controls. This unexpected result may reflect complex regulation of Bcl-2 transcription, suggesting that cyclin E may be interacting with multiple regulatory factors. We are in the processes of identifying the mechanisms that govern Bcl-2 regulation by cyclin E. In both MCF7 and T47D cyclin E overexpressing cells,

the levels of Bad and Bax are increased. Therefore, in both cell lines, cyclin E overexpression alters the regulation of three proteins important for apoptosis. The modulation of the pro- and antiapoptotic proteins is dependent on the extent of cyclin E overexpression and on the cellular context. In this study we address the consequences of deregulation of the pro and antiapoptotic proteins. The signaling pathways for TNF-a, Fas, and TRAIL have been well characterized. Bcl-2 overexpression in MCF7 cells has been shown to inhibit TNF-a, Fas, and TRAILmediated apoptosis. Therefore, we hypothesized that a decrease in Bcl-2 and a small increase in Bax and Bad alter the ratio of pro- and antiapoptotic proteins to make the cells more susceptible to these agents. In MCF7 cells, the greatest increase in TRAIL sensitivity is observed in cells that express the highest amount of cyclin E and have the greatest decrease in the level of Bcl-2. The response to Fas and TNF-a is also enhanced in cyclin E overexpressing cells but to a lesser extent. This suggests that a deregulation of pro- and antiapoptotic proteins is a component of heightened sensitivity to cytokines. T47D-CycE 18 cells, which have the highest increase in cyclin E expression but also have an elevation rather than decrease of Bcl-2, are more sensitive to TRAIL than control cells. This suggests that the elevation of cyclin E even in the presence of Bcl-2 sensitizes cells to this cytokine. One plausible explanation is that cyclin E overexpressing cells have higher levels of TRAIL receptors. However, we found that the levels of the TRAIL receptors DR5 and DR4 do not change (Figure 1d); therefore, enhanced sensitivity is not because of an increase in receptor levels. In these cells, the enhanced sensitivity may be because of the increase in the levels of the proapoptotic proteins Bax and Bad, and this increase may be sufficient to compensate for the increase in Bcl-2. Alternatively, cyclin E expression may be modifying other signaling pathways that regulate apoptotic responses. Previous studies have shown that T47D sensitivity to Fas is because of an increase in Fas receptors.13 Even at reduced concentrations of Fas, over 50% of the control cells underwent apoptosis. Since these cells are already very sensitive to Fas, it is not surprising that cyclin E overexpression did not further augment sensitivity to this cytokine. A number of recent studies are premised on the possibility of sensitizing cells to undergo apoptosis by reducing the levels of the antiapoptotic proteins, such as Bcl-2 and Bcl-XL.36–40 In cell culture, antisense oligonucleotides have been used to block the expression of Bcl-2 and sensitize cells to chemotherapy.36–40 These studies demonstrate that altering the homeostasis of pro- and antiapoptotic proteins can facilitate cell death, suggesting a potential therapeutic application of Bcl-2 antisense oligonucleotides in the treatment of cancer. However, our study suggests that multiple factors, such as the ratio of pro- and antiapoptotic proteins, the expression of cell cycle regulatory proteins and cellular context, can regulate apoptosis. This is the first study to report an enhanced sensitivity of cyclin E overexpressing cells to cytokine-mediated apoptosis. TNF-a and Fas are not suitable for anticancer therapy since systemic introduction of these agents is severely toxic.41 In contrast, systemic administration of

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TRAIL does not result in toxicity when tested in mouse and non-human primate models.27,41–44 In addition, in mouse studies, systemic administration of TRAIL results in a regression of tumors,27 strongly suggesting that TRAIL is a promising anticancer agent. It is unclear why TRAIL preferentially targets transformed cells; however, this property can be exploited in chemotherapy. Transformed cells have multiple genetic and biochemical alterations, including alterations of cell cycle components. Cyclin E overexpression is a feature of many tumors and has been well studied in breast cancer. Overexpression of this cyclin is associated with a resistance to both chemotherapeutics and radiation, hence it correlates with a poor clinical outcome. In our study, cyclin E overexpression in two different cell backgrounds sensitizes cells to TRAIL-mediated apoptosis. Further studies, including studies of animal models and other cell lines, will be required to determine if the link between cyclin E overexpression and sensitivity to TRAIL is restricted to specific cellular backgrounds or is a universal feature of cyclin E overexpression.

Materials and methods Cell lines The MCF7 cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and propagated in RPMI (Gibco/BRL, Rockville, MD, USA) supplemented with 5% FBS, penicillin/streptomycin (P/S) at 371C and 5% CO2. Cyclin E overexpressing lines were generated by transfection with the pRcCMV-cyclin E plasmid. The control vector pRcCMVneo was obtained from Invitrogen (Carlsbad, CA, USA). The pRcCMVcyclin E plasmid was a gift from Dr P Hines. After transfection, the cells were placed in media containing 400 mg/ml of geneticin (Gibco/BRL), and individual colonies were isolated and propagated in media containing 400 mg/ml geneticin. Immunoblot analysis Cells were lysed in RIPA buffer and 30 mg of total protein was separated on SDS-PAGE gels. Each gel was transferred to BA-85 membrane (Schleicher and Schuell, Keene, NH, USA) and the membrane was stained with Ponceau S to ensure equal loading of protein. The membrane was blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) and 0.1% Tween for 1 h before the addition of specific antibodies. The following antibodies were used: cyclin E antibody (generous gift from Dr G Lozano), Bcl-2 (Santa Cruz, CA, USA), Bcl-XL (Santa Cruz), Bax (Santa Cruz), Bad (Santa Cruz), Mcl-1 (Santa Cruz), and Actin (Santa Cruz). After washing and incubation with secondary mouse monoclonal or rabbit polyclonal horseradish-peroxidaselinked NIF 825 antibodies (Amersham Pharmacia, Piscataway, NJ, USA) proteins were detected using ECL (Amersham Pharmacia) following the manufacturer’s recommendations. Image Quant V.3.0 (Molecular Dynamics, Sunnyvale, CA, USA) software was used to quantitate protein levels. In vitro kinase assays In vitro kinase assays were performed as previously described.45 Briefly, cells were lysed in NP40 lysis buffer, Genes and Immunity

the extracts were precleared and incubated with specific antibodies. The antibody/protein complexes were isolated on Protein A/Protein G agarose beads (Oncogene Science, Cambridge, MA, USA), washed, and resuspended in kinase buffer (1 mg/ml histone H1, 1 mM ATP, 1 mCi/ml g32P ATP, 20 mM HEPES pH 7.0, 80 mM bglycerolphosphate, 20 mM EGTA, 50 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 2.5 mM PMSF, 10 mM camp protein kinase inhibitor). The kinase reaction was terminated by the addition of gel loading buffer and the protein was separated on 12% SDS-PAGE. The gel was fixed, dried, and autoradiographed. Trypan blue exclusion assay Cells were plated at a density of 5 104 cells/ml in 35 mm dishes and allowed to grow overnight. For the induction of Fas-mediated apoptosis, 250 ng/ml anti-Fas (Upstate Biotechnology, Waltham, MA, USA) and 10 mg/ ml cyclohexamide (Sigma, St Louis, MO, USA) for MCF7 cells or 125 ng/ml anti-Fas for T47D cells was added to each dish and the cells were incubated for 24 h. For the induction of TNF-a-mediated apoptosis, 100 ng/ml TNFa (R&D Systems, Minneapolis, MN, USA) was added to each dish and the cells were incubated for 24 h. For the induction of apoptosis by TRAIL, 50 ng/ml of TRAIL was added to each dish and the cells were incubated for 24 h. After incubation, the amount of apoptosis was quantitated by the trypan blue exclusion assay. Briefly, media containing floating cells are first collected into a centrifuge tube. The remaining adherent cells are then trypsinized and added to the collected media. After centrifugation, the cells are resuspended in an equal volume of 0.4% trypan blue (Gibco BRL). After 5 min incubation at room temperature, cells are counted using a hemoacytometer. All results are presented as the percentage of apoptotic cells (ratio of blue stained cells vs the total number of cells). TUNEL assay Cells are plated at a density of 5 104 cells/ml in 35 mm dishes and grown in complete media overnight. MCF7 cells were exposed to 100 ng/ml TNF, 250 ng/ml Fas, and 10 mg/ml cyclohexamide, or 50 ng/ml TRAIL and incubated for 24 h. T47D cells were exposed to 100 ng/ml TNF, 125 ng/ml FAS, or 50 ng/ml TRAIL for 24 h. Media containing floating cells were then transferred into a centrifuge tube. Adherent cells were then trypsinized and transferred into the media containing the floating cells. The cells were centrifuged at 1200 rpm for 5 min, rinsed once with 1 PBS, spotted onto slides, and airdried. The TUNEL assays were performed according to the manufacturer’s instruction (Promega). Mounting solution of 20 ml (0.5 mg/ml PI, 2M DABCO, 50% glycerol) was applied to each slide and a coverslip was added. Cell analysis was performed using a laser scanning cytometer (CompuCyte Corp, Cambridge, MA, USA)46 and fluorescence was measured as described.47

Acknowledgements We thank Dr Gigi Lozano for the anticyclin E antibodies and Dr Janine LaSalle for help with laser scanning cytometry. A VA Merit Award (MM) and a UC Davis Health Systems Award (MM) supported this research.


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