Bcl-2-Overexpressing Cells Caspase-independent ...

Unsymmetrically Substituted Polyamine Analogue Induces Caspase-independent Programmed Cell Death in Bcl-2-Overexpressing Cells Hyo Chol Ha, Patrick M. Woster and Robert A. Casero, Jr. Cancer Res 1998;58:2711-2714.

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[CANCER RESEARCH 58, 2711-2714,

July 1. 1998]

Advances in Brief

Unsymmetrically Substituted Polyamine Analogue Induces Caspase-independent Programmed Cell Death in Bcl-2-Overexpressing Cells1 Hyo Choi Ha, Patrick M. Woster, and Robert A. Casero, Jr.2 The Oncology Center Research Laboratories, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231 Â¡H.C. H., R. A. C.]: Division of Toxicological Sciences, Department of Environmental Health Sciences, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205 [H. C. H., R. A. C.]: and Wayne State University. Detroit, Michigan 48202 [P. M. W.Â¡

drial function, activation of a cell death protease, and DNA and nuclear fragmentation.

Abstract The polyamine analogue, A'1-ctliyl-.V"-|U'vcloheptyl(methyl|-4,K-dia/,aundecane (CHENSpm)-induced programmed cell death in NCI H157 cells is accompanied by cytochrome c release, the loss of mitochondria) mem brane potential, activation of caspase-3, caspase-mediated poly(ADPribose) polymerase cleavage, G2-M arrest, and DNA and nuclear frag mentation. Overexpression of Bcl-2 completely inhibits CHENSpminduced cytochrome c release, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. However, Bcl-2 does not abrogate CHENSpm-

Materials and Methods Materials. CHENSpm was synthesized and used as reported previously (8). Proteinase K and RNase A were purchased from Life Technologies, Inc. Cell Culture and Transfection of the bcl-2 Gene. NCI HI57 cells were maintained as reported previously (9). NO HI57 cells were stably transfected with pCEP4 vector containing the human bcl-2 gene or the hygromycin B

induced programmed cell death. These results suggest that although cytochrome c release and activation of the caspase-3 protease cascade contribute to the rapid and efficient execution of apoptosis, a caspase cascade-independent pathway also exists and can be activated by CHENSpm treatment.

Introduction The polyamines spermidine and spermine and their diamine pre cursor putrescine are cationic molecules found in all eukaryotic cells and are essential for cell proliferation (1,2). High activity of ornithine decarboxylase, a rate-limiting enzyme in polyamine synthesis, and increased levels of intracellular polyamines are known to occur in rapidly proliferating cells or cells undergoing differentiation and transformation (3). Thus, polyamine metabolism is a tempting chemotherapeutic target. One approach has been to develop polyamine analogues that are structurally similar to the natural polyamines in their regulation of polyamine metabolism but not in their growth and survival functions. Our previous studies demonstrated that treatment with 10 /AMCHENSpm,3 a polyamine analogue, induced PCD with HMW DNA fragmentation and G2-M arrest in NCI HI57, a human non-small cell lung carcinoma cell line (4). The family of cysteinyl aspartate-specific proteinases (caspases) mediates a highly specific proteolytic cleavage early in the process of apoptosis (5). Mitochondria! dysfunction, such as loss of mitochon dria! transmembrane potential, has also been observed in the early stages of apoptosis (6). Recently, cytochrome c release from the mitochondria to the cytosol has been shown to activate caspase-3, thus continuing transmission of the apoptotic program (7). In an attempt to better characterize the mechanism of CHENSpm-induced PCD, we examined the effects of overexpression of the antiapoptotic protein Bcl-2 in CHENSpm-treated NCI HI57 cells by analyzing mitochonReceived 3/13/98; accepted 4/30/98. 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. 'This work was supported in part by Grants ES07141, CA57545, CA63552, CA58184, andCA5l085. 2 To whom requests for reprints should be addressed, at Johns Hopkins Oncology Center Research Laboratories, 424 North Bond St., Baltimore, MD 21231. Phone: (410)955-8580; Fax: (410)614-9884; E-mail: [email protected]. 'The abbreviations used are: CHENSpm, W'-ethyl-A'"-[(cycloheptyl)methyl]4,8diazaundecane; HMW, high molecular weight; PARP. poly(ADP-ribose) polymerase; PCD, programmed cell death; TÃšNEL, deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

(HPH) resistance gene only (obtained from Dr. Bert Vogelstein, Johns Hop kins University School of Medicine; Ref. 10). Polyamine pools were deter mined as reported previously (9) and are generally reproducible within 15%. Cell Cycle Analysis and Assessment of Morphology. Flow cytometric and morphological analyses of cells were performed as described previously (11). Analysis of Apoptosis. Apoptosis was assessed using the in situ cell death detection kit (Boehringer Mannheim, Indianapolis, IN) according to the man ufacturer's protocol. Mitochondria! Membrane Potential and Cell Viability Assays. Adher ent and nonadherent cells, CHENSpm-treated and untreated, were harvested and incubated in 15 Â¿tM Rhl23 (Molecular Probes, Eugene, OR) for 15 min at 37Â°Cfor measurement of mitochondria! membrane potential as reported (12). Cells were then washed twice and resuspended in PBS and analyzed on a Becton Dickinson FACScan. Cell viability was assessed by trypan blue ex clusion. Western Blot Analysis. Mitochondrial and cytosolic (S-100) fractions were prepared as published previously (13). Antibodies against the following proteins were purchased from the indicated suppliers: human CPP32 from Transduction Laboratories (Lexington, KY), PARP from Boehringer Mann heim (Indianapolis, IN), human bcl-2 from DAKO (Carpinteria, CA), ÃŸ-actin from Sigma (St. Louis, MO), and cytochrome c from PharMingen (San Diego, CA). Cell lysates from control and CHENSpm-treated cells were resolved by SDS-PAGE and transferred to nitrocellulose. Immunoblot analysis was per formed with the Enhanced Chemiluminescence (ECL) Western blotting detec tion reagents from Amersham (Buckinghamshire, England).

Results Effects of Bcl-2 on CHENSpm-induced Cell Death. The expres sion of endogenous antiapoptotic protein Bcl-2 was undetectable in NCI HI57 cells (Fig. 1A). To evaluate the role of Bcl-2 on CHENSpm-induced PCD, NCI HI57 cells were transfected with the human Bcl-2 gene (NCI H157/ÃŸc/-2) or an empty vector control (NCI H\51IHPH). Overexpression of Bcl-2 in NCI HI57 cells was con firmed by protein immunoblot analysis using an antibody to Bcl-2 (Fig. 1A).Overexpression of Bcl-2 modestly increased cell viability in NCI HI57 cells treated with CHENSpm compared with controls (Fig. IB). However, overexpression of Bcl-2 did not prevent but only delayed CHENSpm-induced cell death. Also, overexpression of Bcl-2 did not significantly alter CHENSpm-treated intracellular polyamine pools or analogue accumulation. The concentrations of putrescine, spermidine, spermine, and CHENSpm in vector control cells were 3.3, 7.2, 12.0, and 10.1 nmol/mg protein, whereas in Bcl-2-overexpressing

2711 Downloaded from cancerres.aacrjournals.org on June 7, 2013. © 1998 American Association for Cancer Research.

CHENSPM-INDUCED

cytoplasm (Fig. 2A). In cells treated with CHENSpm, the amount of cytochrome c in the mitochondria was decreased concurrent with the increase of cytochrome c in the cytoplasm. In untreated cells, the level and location of cytochrome c were unchanged in both vector control and Bcl-2-overexpressing (Fig. 2A) cells. Importantly, the overexpression of antiapoptotic protein Bcl-2 in NCI H157 cells completely blocked CHENSpm-induced release of cytochrome c from the mito chondria into the cytoplasm (Fig. 2A). To determine whether the release of mitochondria! cytochrome c is associated with the mito chondria! membrane depolarization, mitochondria! membrane poten tial was assessed by Rhl23 fluorescence. Rhl23 is a cell-permeable cationic fluorescent dye and is accumulated by mitochondria, depend ing on the membrane potential. A significant number of vector control cells treated with CHENSpm for 24 h were unable to accumulate Rhl23 as compared with untreated control cells (Fig. 2B), indicating a decrease in mitochondria! membrane potential. Interestingly, the overexpression of Bcl-2 in NCI H157 only partially prevented CHENSpm-induced loss of mitochondria! membrane potential as compared with untreated control Bcl-2 cells (Fig. 25). Therefore, the release of cytochrome c does not appear to be coupled to the loss of mitochondria membrane potential because Bcl-2-overexpressing cells did not maintain a mitochondria! membrane potential after treatment with CHENSpm. Effects of Bcl-2 on CHENSpm-induced Activation of Caspase-3 and Cleavage of PARP. Treatment of vector control cells with CHENSpm for 24 h resulted in cleavage and a decline in caspase-3 levels, hence activating an intracellular apoptotic signal by proteolysis (Fig. 3/1). Activated caspase-3 is known to cleave substrates including PARP. Treatment with CHENSpm for 24 h resulted in the cleavage of

Btl-2

B-actin HPH

PCD

Bcl-2

Bcl-2

HPH

DNA Content Fig. 1. The effects of overexpression of Bcl-2 in CHENSpm-induced cell death and G2-M arrest in NCI H157 cells. In A, the overexpression of Bcl-2 was confirmed by protein immunoblot analysis using an antibody to Bcl-2. In B, NCI H157 and Bcl-2overexpressing NCI HI57 cells were treated with 10 /Â¿MCHENSpm and assessed for viability by trypan blue exclusion in three independent experiments. The percentage of viable cells was calculated relative to untreated cells. C, flow cytometric analysis of NCI H157 cells. Adherent and nonadherent cells after treatment of 24 h were harvested and stained with propidium iodide. Cell number and DNA content are represented by the ordinate and abscissa, respectively. The G|. S. and G2-M fractions were shaded and quantitated by using the MULTICYCLE software package. Vector control cells (NCI HÃŒ51/HPH):37.9% G,, 50.2% S. and 12.0% G,-M; Bcl-2-overexpressing cells (NCI H157/Ã„C-/-2):32.9% Gâ€ž47.1% S, and 20.1% G,-M; vector control cells treated with 10 /XMCHENSpm: 23.7% G,, 15.9% S. and 60.4% G2-M; and Bcl-2-overexpressing cells (NCI H157/ÃŸd-2) treated with 10 Â¿unCHENSpm: 13.2% G,, 46.5% S, and 40.4% G2-M.

Cytosolic cytochrome c

Mitochondria! cytochrome c Bcl-2

HPH

cells, the concentrations were 3.4, 8.1, 17.6, and 10.4 nmol/mg pro tein, respectively. Effects of bcl-2 on CHENSpm-induced G2-M Arrest. Treatment of vector control NCI H157/HPH cells with CHENSpm for 24 h induced G2-M arrest (Fig. 1C); -60% of cells treated with CHENSpm were in G2-M, whereas ~ 12% of cells in control were in G2-M. Also, treatment with CHENSpm decreased the number of S phase cells to 16%, whereas â€”¿50% of control cells were in S phase. Treatment of NCI H157/ÃŸc/-2cells with CHENSpm for 24 h also induced G2-M arrest; â€”¿40% of cells treated with CHENSpm were in G2-M, whereas â€”¿20% of cells in the untreated control were in G2-M. The population of S phase cells in Bcl-2 transfectants were maintained at 47% for both treated and untreated cells. Effects of Bcl-2 on CHENSpm-induced Release of Cytochrome c from Mitochondria and Loss of Mitochondria! Membrane Po tential. Treatment of vector control cells with CHENSpm for 24 h resulted in the release of cytochrome c from the mitochondria into the

HPH

Bcl-2 Rhodimlne

123

Fig. 2. The effects of Bcl-2 on CHENSpm-induced release of cytochrome c from mitochondria into the cytoplasm and loss of mitochondria! membrane potential in NCI H157 cells. A, immunoblot analysis of cytochrome c in S-IOO (cytosolic) fraction and mitochondria isolated from CHENSpm-treated or -untreated NCI H157/HPH cells and NCI H157/Bcl-2 cells for 24 h. fi, control cells and Bcl-2-overexpressing cells with or without 10 fiM CHENSpm treatment for 24 h. Adherent and nonadherent cells, treated or not treated, were harvested and incubated with Rhl23. The percentage of cells falling within the range of Rhl23 fluorescence indicative of depolarized cells is shown.

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CHENSPM-INDUCED

does not appear to be absolutely required for activation of caspase-3 or induction of apoptosis in all systems (14, 15). Overexpression of Bcl-2 has been demonstrated to prevent the activation of caspase-3 by acting both upstream (13) and downstream

I

(16) from cytochrome c release in some systems. However, NCI HI57 cells overexpressing Bcl-2 treated with CHENSpm still die without

Caspase-3

p-actin

HPH

â€”¿ 116kDaPARP HPH

â€”¿85 kDa fragment

Bcl-2

â€”¿116 kDa PARP

24 h

PCD

48 h

Fig. 3. The effects of Bcl-2 on CHENSpm-induced caspase-3 degradation and PARP cleavage in NCI H157 cells (NCI H\51IHPH). In A. immunoblot analysis of caspase-3 was performed on extraÃ©isfrom CHENSpm-treated or -untreated NCI H157/WH and NCI H157/ÃŸr/-2. Treated cells were exposed to 10 JÃ•M CHENSpm for 24 h. The results indicate that the overexpression of Bcl-2 completely inhibits CHENSpm-induced caspase-3 degradation. In B. complete inhibition of CHENSpm-induced PARP cleavage by the overexpression of Bcl-2 was confirmed by immunoblot analysis of PARP from CHENSpm-treated or -untreated NCI H157/H/VÃ• and NCI H157/ÃŸr/-2 cells. Where indicated, cells were treated with 10 Â¿IM CHENSpm for 24. 36. and 48 h.

measurable cytochrome c release or caspase activation. These data suggest that cytochrome c release and the caspase-3 protease cascade contribute to the rapid and efficient apoptotic process but are not absolutely necessary to the commitment of all apoptotic processes in CHENSpm-treated NCI HI57 cells. The existence of a caspase-3independent apoptotic pathway has also been demonstrated in caspase-3-deficient mice, where thymocytes have been shown to retain normal susceptibility to various apoptotic stimuli, suggesting system redundancy (17). The results of studies with the caspase-3 knockout mice (17) demonstrate that the activation of the caspase cascade, although sufficient in some systems for the induction of the apoptotic pathway, is not an absolute requirement. Furthermore, the possibility that CHENSpm acts directly on events downstream or aside from Bcl-2 function cannot presently be excluded. In summary, our results suggest that multiple parallel and possibly independent apoptotic pathways exist in NCI HI57 cells, rather than a single linear pathway. Overexpression of Bcl-2 completely prevents cytochrome c release and activation of the caspase-3 initiated cascade but does not abrogate CHENSpm-induced PCD. Cytochrome c and caspases have been shown to play pivotal roles in apoptotic processes in many cases; however, cytochrome c and caspases are not absolutely required for all apoptotic processes. This notion may explain why Bcl-2 cannot

PARP with the appearance of the expected Mr 85,000 apoptotic fragment in NCI H157/HPH (Fig. 3ÃŸ).By contrast, the overexpres sion of Bcl-2 completely inhibited CHENSpm-induced activation of caspase-3 and cleavage of PARP up to 48 h after the treatment, where more than 80% of cells were dead (Figs. \A, 3A, and 3fl). Effects of Bcl-2 on CHENSpm-induced Nuclear and DNA Frag mentation. Our previous studies indicated that treatment with CHENSpm for 24 h resulted in an induction of HMW DNA fragmen tation (2:50 kb) as detected by field-inversion gel electrophoresis. Single- and double-stranded DNA breaks, which are clearly evident 24 h after treatment with CHENSpm, were detected by TÃšNEL (Fig. 4A). Furthermore, the formation of apoptotic nuclei (condensed or fragmented) was observed with CHENSpm treatment at 24 h (Fig. 4ÃŸ).Surprisingly, the overexpression of Bcl-2 had only a modest effect on the formation of HMW DNA fragmentation (data not shown), single- and double-stranded DNA breaks, and the formation of apoptotic nuclei observed in cells treated with CHENSpm (Fig. 4). As measured in the TÃšNEL assay, Bcl-2 reduced the fluorescent label incorporation from 72 to 48%. Thus, the overexpression of Bcl-2 in

48% 1IPI1

Bcl-2

Fluorescence Intensity

NCI HI57 only reduced or delayed the formation of DNA and nuclear fragmentation but was not capable of preventing it. Discussion We have demonstrated previously that induction of PCD in NCI H157 cells by certain polyamine analogues was, in part, the result of the production of H2O2 generated through polyamine catabolism (7). Although CHENSpm treatment does not result in a significant induc tion of polyamine catabolism or subsequent H2O2 production, it is as effective in producing PCD as the H2O2-inducing analogues. The release of cytochrome c from the mitochondria to the cyto plasm activates caspase-3, continuing the transmission of the apop totic signals (3), and microinjection of cytochrome c induces apopto sis in cells expressing pro-caspase-3. However, cytochrome c release

Fig. 4. The effects of CHENSpm-induced DNA and nuclear fragmentation in NCI HI57 cells. In A. control cells and Bcl-2-overexprcssing cells were treated with 10 JÂ¿M CHENSpm for 24 h. After indicated treatment, adherent and nonadherent cells were harvested and incubated with TÃšNEL reaction mixture according to the manufacturer's suggestion. The percentage of cells falling within the range of dUTP fluorescence indicative of apoptotic cells is shown. In B. control cells and Bcl-2-overexpressing cells were treated with 10 Â¿AM CHENSpm for 24 h. Adherent and nonadherent cells were harvested and fixed with Hoechst dye. visualized under UV excitation, and photographed with a Nikon Labophot microscope. The field illustrated is representative of the entire population observed.

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CHENSPM-INDUCED

inhibit but only delay apoptosis in some situations. CHENSpm, a polyamine analogue synthesized as an antineoplastic agent, induces cytotoxicity through a mechanism that leads to DNA damage concurrent with G2-M arrest in a p53-independent manner. Therefore, it is significant that CHENSpm is capable of inducing PCD in a caspase-3-independent manner in cells overexpressing antiapoptotic protein Bcl-2.

7. Liu, X., Kim, C. N., Yang. J.. Jemmerson. R., and Wang, X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell, 86: 147-157, 1996. 8. Wosler, P. M. S-Adenosylmethionine decarboxylase and spermidineyspermine-N1-

9.

Acknowledgments We thank Drs. Kornelia Polyak, J. Marie Hardwick, James D. Yager, John T. Isaacs, and Bertrand Tombal for helpful discussions and Jim Flook for expert technical assistance with fluorescence-activated cell sorting analysis.

10.

11.

We are grateful to Tracy R. Murray for help in preparing the manuscript. 12.

References 1. Pegg. A. E. Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res., 48: 759-774, 1988. 2. Marlon, L. J., and Pegg, A. E. Polyamines as targets for therapeutic intervention. Annu. Rev. Pharmacol., 35: 55-91, 1995. 3. JÃ¤nne,J., Alhonen, L., and Leinonen, P. Polyamines: from molecular biology to clinical applications. Ann. Med.. 23: 241-259. 1991. 4. Ha, H. C., Woster, P. M., Yager, J. D., and Casero, R. A., Jr. The role of polyamine catabolism in polyamine analogue-induced programmed cell death. Proc. Nati. Acad. Sci. USA, 94: 11557-11562. 1997. 5. Nicholson, D. W., and Thornberry, N. A. Caspases: killer proteases. Trends Biochem. Sei., 22: 299-306. 1997. 6. Zamzami, N.. Marcheni, P., Castedo, M., Zanin, C.. Vayssiere, J-L., Petit, P. X., and Kroemer, G. Reduction in mitochondria! potential constitutes an early irreversible step of programmed lymphocyte death in vivo. I. Exp. Med., 1K1: 1661-1672. 1995.

PCD

13.

14.

15. 16.

17.

acetyltransferase emerging targets for rational inhibitor. In: R. A. Casero, Jr. (eds.), Polyamines: Regulation and Molecular Interaction, pp. 171-186. Austin: R. G. Lander Co.. 1995. Casero, R. A., Ervin, S. J., Celano, P., Baylin, S. B., and Bergeron. R. J. Differential response to treatment with the bis(ethyl)polyamine analogues between human small cell lung carcinoma and undifferentiated large cell lung carcinoma in culture. Cancer Res., 49: 639-643, 1989. Pietenpol, J. A., Papadopoulos, N., Markowitz, S., Willson, J. K. V.. Kinzler, K. W., and Vogelstein, B. Paradoxical inhibition of solid tumor cell growth by bcl2. Cancer Res., 54:3714-3717, 1994. Dietch, A. D., Law, H., and de Vere White, R. A stable propidium iodide staining procedure for flow cytometry. J. Histochem. Cytochem., 30: 967-972, 1982. Vander Heiden, M. G., Chande!, N. S., Williamson, E. K., Schumacker, P. T., and Thompson, C. B. Bcl-XL regulates the membrane potential and volume homeostasis of mitochondria. Cell, 91: 627-637, 1997. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado. A. M., Cai, J., Peng, T-L, Jones, D. P.. and Wang, X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science (Washington DC), 275: 1129-1132, 1997. Li, F.. Srinivasan, A.. Wang. Y., Armstrong, R. C., Tornaseli!, K. J., and Fritz, L. C. Cell-specific induction of apoptosis by microinjection of cytochrome c. J. Biol. Chem.. 272: 30299-30305. 1997. Zhivotovsky, B.. Orrenius, S., Brustugun, O. T.. and D0skeland, S. O. Injected cytochrome c induces apoptosis. Nature (Lond.), 391: 449-450, 1998. RossÃ©,T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B., and Bomer. C. Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature (Lond.). 391: 496-499. 1998. Kuida, K., Zheng, T. S., Na, S., Kuan, C., Yang, D., Karasuyama, H., Rakic, P., and Flave!!, R. A. Decreased apoptosis in the brain and premature lethality in CPP32deficient mice. Nature (Lond.), 384: 368-372. 1996.

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